Food Control 21 (2010) 12821290

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Preventive effect of tannic acid in combination with modied atmospheric

packaging on the quality losses of the refrigerated ground beef
Sajid Maqsood, Soottawat Benjakul *
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Chemical, microbiological and sensorial changes of ground beef treated without and with tannic acid
Received 20 November 2009 (200 mg/kg) and stored in air and under modied atmosphere packaging (MAP) (80%O2/20%CO2 or
Received in revised form 19 February 2010 10%O2/20%CO2/70%N2) were monitored during 15 days of storage at 4 C. During the storage, samples
Accepted 27 February 2010
treated with tannic acid and kept under all packaging conditions contained lower peroxide value (PV)
and thiobarbituric acid-reactive substances (TBARS) with coincidental lower non-haem iron content,
compared with non-treated counterparts (P < 0.05). The sample packed in high oxygen MAP treated with-
Keywords:
out and with tannic acid had the higher oxymyoglobin and a values and received the higher likeness
Lipid oxidation
MAP
scores for colour, whereas the samples stored in air and under low oxygen MAP showed the lower values,
Ground beef regardless of tannic acid treatment. After 15 days of storage, myosin heavy chain (MHC) and actin of all
Colour tannic acid treated samples underwent less degradation than those without tannic acid treatment for all
Tannic acid packaging conditions. Degradation of MHC was more pronounced in samples kept under MAP with high
Quality oxygen. Psychrophilic bacterial count (PBC) of all tannic acid treated samples was lower, compared with
that of non-treated samples (P < 0.05), irrespective of packaging condition. Therefore, tannic acid treated
samples stored under high oxygen MAP could maintain the red colour and retard lipid oxidation and
microbial growth of ground beef during refrigerated storage.
2010 Elsevier Ltd. All rights reserved.

1. Introduction tannic acid exhibited the superior radical scavenging activities as

well as reducing power and effectively inhibited the lipid oxidation
The colour and odour of meat are the most important quality in sh mince and emulsion model systems. Tannic acid is afrmed
attributes for the consumers by which meat quality is readily as- as Generally Recognized as Safe (GRAS) by the Food and Drug
sessed (Mancini & Hunt, 2005). Myoglobin (Mb) is the principle Administration (FDA) for the use as a direct additive in some food
haem protein responsible for meat colour. The oxygenated form products including meat products (21 CFR184. 1097, U.S. Code of
of Mb or oxymyoglobin (OxyMb) contributes to the cherry red col- Federal Regulations, 2006). Thus, the objective of the present study
our associated with fresh meat (Bou et al., 2008). However, OxyMb was to investigate the combined effect of tannic acid and high- and
can change its colour to brown due to the formation of metmyoglo- low-oxygen modied atmospheric packaging on the prevention of
bin (MetMb) (Bou et al., 2008). lipid oxidation and colour stability in the ground beef.
For meat, modied atmosphere packaging with a high level of
oxygen (7080%) is required to maintain the acceptable red colour.
2. Materials and methods
High oxygen atmosphere preserves the bright red colour of meat
(Okayama, Muguruma, Murakami, & Yamada, 1995). Although
2.1. Chemicals
high oxygen MAP maintains the redness of meat during storage,
the rancidity often develops (Jayasingh, Cornforth, Brennand, Car-
All the chemicals and reagents were of analytical grade and
penter, & Whittier, 2002; Okayama et al., 1995). To alleviate such
were purchased from Sigma Chemical Co. (St. Louis, MO, USA),
a drawback, the use of antioxidants, especially phenolic com-
Merck (Darmstadt, Germany), Fluka Chemical Co. (Buchs, Switzer-
pounds could be an effective means to prevent lipid oxidation in
land) and Bio-Rad (Richmond, CA, USA).
high oxygen packaging (Morrissey, Sheehy, Galvin, Kerry, & Buck-
ley, 1998). Recently, Maqsood and Benjakul (2010a) reported that
2.2. Sample preparation

* Corresponding author. Tel.: +66 7428 6334; fax: +66 7421 2889. Fresh beef tendon loin (5 kg) was purchased from a local
E-mail address: soottawat.b@psu.ac.th (S. Benjakul). slaughter house in Hat Yai, Songkhla, Thailand. The beef was

0956-7135/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.02.018
 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290
packed in polythene bags and kept in ice during transportation to
Department of Food Technology, Prince of Songkla University.
Upon arrival, the sample was rinsed with the chilled sterilized dis-
tilled water (810 C), cut into small pieces and the connective tis-
sue and depot fat were removed manually. The prepared sample
was minced using a mincer with the hole diameter of 3 mm.
Ground beef was packed in polythene bags and kept at 4 C until
use but was not longer than 2 h.
2.3. Tannic acid treatment and modied atmosphere packaging (MAP)
of ground beef
Ground beef was divided into six portions of 800 g each. Tannic
acid (0.16 g) was dissolved in 25 ml of sterilized distilled water and
pH was adjusted to neutral by 2 M NaOH. Three portions of ground
beef (800 g) were separately added with 25 ml of neutralized solu-
tion of tannic acid to obtain a nal concentration of 200 mg/kg.
Other three portions without addition of tannic acid were used
as control and the same volume of sterilized distilled water was
added instead. The ground beef (60 g) treated without and with
tannic acid was placed on the polystyrene trays (20  12 cm2)
and inserted in the nylon/LLDPE bag (30  16 cm) (Asian Foams,
Hat Yai, Thailand) with the thickness of 0.08 mm and gas perme-
ability (CO2, N2 and O2: 1.7  1010, 0.1  1010 and 0.4 
1010 m3 mm/cm2 s cm Hg at 25 C, 1 atm pressure, respectively)
and was packed with a sample/gas ratio of 1:3 (w/v) using a Hen-
kovac type 1000 (Tecnovac, Italy). Two gas mixtures containing
10%O2/20%CO2/70%N2 or 80%O2/20% CO2 with a pressure of 5 kg/
cm2 were lled in the bag. Ground beef treated without and with
tannic acid and packed in air were designated as A1(T) and
A1(+T) respectively. Those packed under MAP with low oxygen
concentration (10%) and added without and with tannic acid were
referred to as M1(T) and M1(+T). Those packed under MAP with
high oxygen concentration (80%) and treated without and with
tannic acid were designated as M2(T) and M2(+T), respectively.
All samples were stored at 4 C and taken for chemical and sensory
analyses every 3 days for 15 days, except sensory analysis was
omitted at day 12. For microbiological analysis and SDSPAGE,
samples were taken for analyses at day 0 and 15.
2.4. Chemical analysis
2.4.1. Peroxide value
Peroxide value was determined as per the method of Maqsood
and Benjakul (2010b). A standard curve was prepared using cu-
mene hydroperoxide at the concentration range of 0.52 ppm. Per-
oxide value was expressed as lmol of peroxide/kg of sample.
2.4.2. Thiobarbituric acid-reactive substances
Thiobarbituric acid-reactive substances (TBARS) were deter-
mined as described by Maqsood and Benjakul (2010a). Standard
curve was prepared using 1,1,3,3-tetramethoxypropane (MAD) at
the concentration ranging from 0 to 10 ppm and TBARS were ex-
pressed as mg of MAD equivalents/kg of sample.
2.4.3. Determination of haem iron and non-haem iron contents
The haem iron and non-haem iron contents were determined
according to the method of Gomez-Basauri and Regenstein
(1992) and Schricker, Miller, and Stouffer (1982), respectively as
described by Maqsood and Benjakul (2010a). The haem and non-
haem iron content was expressed as mg/100 g sample.
2.4.4. Determination of oxymyoglobin and metmyoglobin contents
Oxymyoglobin and metmyoglobin contents of ground beef were
determined as described by Carlez, Veciana-Nogues, and Cheftel
(1995) with a slight modication. Ground beef (2 g) was trans-
1283
ferred into a 50 ml-polypropylene centrifuge tube and 20 ml of
cold 40 mM phosphate buffer (pH 6.8) were added. The mixture
was homogenised at a speed of 13,500 rpm for 10 s with a
Ultra-Turrax T25 homogeniser (Janke & Kunkel, Staufen, Germany).
The homogenate was centrifuged using a RC-5B plus centrifuge
(Beckman, JE-AVANTI, Fullerton, CA, USA) at 3000 for 30 min at
4 C. The supernatant was ltered with Whatman No.1 lter paper
(Whatman International, Ltd, Maidstone, England) and the volume
was brought up to 25 ml with the same phosphate buffer. Absor-
bance of the clear supernatant was measured at 525, 545,
565 and 572 nm using a UV-1601 spectrophotometer (Shimadzu,
Kyoto, Japan). The oxymyoglobin (OxyMb) and metmyoglobin
(MetMb) contents were calculated according to Krywicki (1982),
using the following equations:
OxyMb % 0:882R1  1:267R2 0:809R3  0:361  100
MetMb % 2:541R1 0:777R2 0:800R3 1:098  100
where R1, R2 and R3 are the absorbance ratio of A572/A525, A565/A525
and A545/A525, respectively.
2.4.5. Colour determination
Colour of the ground beef samples was measured using a colou-
rimeter (Hunter Lab., Model colour Flex, Reston, VIRG, USA) with
the port size of 0.50 in. The determination of colour was done at
six different positions of the sample. Standardization of the instru-
ment was done using a black and white Minolta calibration plate.
The values were reported in the CIE colour prole system as L-va-
lue (lightness), a-value (redness/greenness), and b-value (yellow-
ness/blueness).
2.4.6. SDSpolyacrylamide gel electrophoresis (SDSPAGE)
Fresh ground beef sample obtained at day 0 and all samples
stored for 15 days were subjected to SDSPAGE according to the
method of Laemmli (1970) as described by Maqsood and Benjakul
(2010b). Quantitative analysis of protein band intensity was per-
formed using a Model GS-700 Imaging Densitometer (Bio-Rad Lab-
oratories, Hercules, CA, USA) with Molecular Analyst Software
version 1.4 (image analysis system). The intensity of interested
protein bands was expressed, relative to that found in fresh sample
at day 0.
2.5. Microbiological analysis
Fresh ground beef (day 0) and all samples stored for 15 days
(25 g) were collected aseptically in a stomacher bag and 10 vol-
umes of sterile saline solution (0.85 g/100 ml) were added. After
homogenising in a Stomacher blender (Stomacher M400, Seward
Ltd., Worthington, England) for 1 min, a series of 10-fold dilutions
was made using normal saline solution (0.85%) for microbiological
analyses. Psychrophilic bacterial counts (PBC) were determined by
plate count agar (PCA) with the incubation at 7 C for 7 days (Cou-
sin, Jay, & Vasavada, 1992). PBC was expressed as log cfu/g.
2.6. Sensory analysis
The sensory evaluation was performed by 30 untrained panel-
ists, who were the graduate students in Food Science and Technol-
ogy programme with the age of 2533 years and were familiar
with beef consumption. The assessment of raw samples was con-
ducted for the colour and odour using a nine-point hedonic scale
(Mailgaad, Civille, & Carr, 1999): 1, dislike extremely; 2, dislike
very much; 3, dislike moderately; 4, dislike slightly; 5, neither like
nor dislike; 6, like slightly; 7, like moderately; 8, like very much; 9,
like extremely. The samples were presented to the panelists just
1284 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290
after opening the packs. The samples were placed in the sterile pet-
ridish and served to panelist.
2.7. Statistical analysis
All experiments were run in triplicate. The experimental data
were subjected to Analysis of Variance (ANOVA) and the differ-
ences between means were evaluated by Duncans New Multiple
Range Test (Steel & Torrie, 1980). For pair comparison, T-test was
used. Data analysis was performed using a SPSS package (SPSS
14.0 for Windows, SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. Effect of tannic acid in combination without and with MAP on the
chemical changes of ground beef during refrigerated storage
3.1.1. Changes in peroxide value (PV) and thiobarbituric acid-reactive
substances (TBARS)
Changes in PV and TBARS of ground beef treated without and
with tannic acid (200 mg/kg) and stored in air and under MAP with
different gas mixtures during refrigerated storage are shown in
Fig. 1a and b, respectively. Gradual increase in PV was found in
all samples throughout the storage period of 15 days (P < 0.05), ex-
cept for the samples without tannic acid treatment, in which the
PV decreased markedly after 9 days of storage (P < 0.05). A de-
crease in PV was most likely due to the decomposition of hydroper-
oxide, a primary oxidation product, to the secondary lipid
oxidation products (Boselli et al., 2005). During the rst 9 days of
storage, the samples without tannic acid treatment underwent a
higher lipid oxidation as evidenced by the higher PV. The oxidation
of those samples more likely proceeded continuously, however the
rate of decomposition of primary oxidation products might be
greater. The samples stored under MAP with high oxygen concen-
A1(+T)
5
(mol of peroxide/kg
4
sample)
3
PV
0
0 3
A1(+T) A1(-T)
14
(mg MAD/kg sample)
12
10
TBARS
0
0 3
Fig. 1. Changes in peroxide value (a) and thiobarbituric acid-reactive substances (b), of ground beef treated without and with tannic acid stored in air and under MAP (10%O2/
20%CO2/70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).
tration without tannic acid treatment [M2(T)] contained the
highest PV, especially within the rst 9 days, while samples treated
with tannic acid and kept under MAP with low oxygen concentra-
tion [M1(+T)] contained the lowest PV (P < 0.05). Tannic acid was
very effective in retarding the propagation stage of lipid oxidation
in ground beef, even when the high oxygen level was used in pack-
aging. Phenolic compounds including tannic acid could retard the
initiation or propagation steps of lipid oxidation reactions by scav-
enging lipid radicals (Maqsood & Benjakul, 2010a) and forming
low-energy antioxidant radicals that do not readily promote oxida-
tion of unsaturated fatty acids.
TBARS values of the ground beef without tannic acid treatment
stored in air and under MAP increased rapidly with increasing stor-
age time (P < 0.05). Among those samples, that stored under MAP
with high oxygen level had the high TBARS values, compared with
others (P < 0.05). For samples treated with tannic acid, the gradual
increase in TBARS was noticeable during the storage. It was noted
that the much lower TBARS values were obtained in samples trea-
ted with tannic acid, regardless of packaging conditions. Jayasingh
et al. (2002) reported that after 6 days of storage, MAP ground beef
samples (80%O2/20%CO2) had higher TBARS than those stored in
air. The higher TBARS were found in ground beef stored under
MAP with increasing oxygen concentrations after 10 days of stor-
age (OGrady et al., 2000). High oxygen partial pressure found in
high oxygen MAP can promote oxidation processes by enabling
oxygen to react with muscle components (OGrady et al., 2000).
From the results, tannic acid treatment effectively retarded the oxi-
dation as shown by no differences in TBARS among all samples
stored under different atmospheres. Lund, Hviid and Skibsted
(2007) found that treatment of beef patties with natural antioxi-
dant (rosemary) retarded the TBARS formation under the MAP with
high oxygen concentration (80%). Grape seed extract and pine bark
extract containing tannic acid signicantly improved the oxidative
stability of cooked beef (Ahn, Grun, & Fernando, 2002). Thus, the
A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)
6 9 12 15
Storage time (days)
(a)
M1(+T) M1(-T) M2(+T) M2(-T)
6 9 12 15
Storage time (days)
(b)
 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290
A1(+T)
8
Haem iron content
(mg/100g sample)
6
0
0 3
A1(+T)
4
Non-haem iron content
(mg/100g sample)
0
0 3
Fig. 2. Changes in haem iron content (a) and non-haem iron content (b) of ground beef treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/
70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).
tannic acid was proved to be an effective antioxidant in the refrig-
erated ground beef packed under high oxygen concentration.
3.1.2. Changes in haem and non-haem iron content
Haem and non-haem iron contents of ground beef treated with-
out and with tannic acid and stored in air and under MAP with dif-
ferent gas compositions during the refrigerated storage are
presented in Fig 2a and b, respectively. Haem iron content of all
ground beef samples decreased gradually as the storage time in-
creased (P < 0.05). Haem pigment concentration in lamb meat de-
creased with increasing storage time (Luciano et al., 2009).
Decrease in haem iron content with increasing storage time was
probably due to the haem breakdown, resulting in the increase in
non-haem iron content (Benjakul & Bauer, 2001). The ground beef
treated with tannic acid and packed under MAP with high oxygen
concentration [M2(+T)] showed the highest haem iron content,
compared with other samples, particularly after 6 days of storage
(P < 0.05). In general, the lower haem iron content was found in
the samples without tannic acid treatment throughout the storage.
Thus, tannic acid might reduce the destruction of haem as indi-
cated by the lowered decrease in haem iron content.
Non-haem iron content of all the samples increased gradually
as the storage time increased (P < 0.05) (Fig 2b). The increase in
non-haem iron content of ground beef was coincidental with the
decrease in haem iron in the ground beef (Fig. 2a). Deterioration
of sub-cellular, organelles, e.g. mitochondria, and the release of
cytochrome-C, could also be responsible for the increase in non-
haem iron (Decker & Hultin, 1990). Decker and Hultin (1990) also
suggested that haem pigment or other iron-containing proteins are
possibly denatured with increasing storage time, resulting in the
release of iron. The sample treated with tannic acid had the lower
non-haem iron content, compared with those without tannic acid
treatment, particularly after 6 days of storage (P < 0.05). The lower
1285
A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)
6 9 12 15
Storage time (days)
(a)
A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)
6 9 12 15
Storage time (days)
(b)
non-haem iron content in the samples treated with tannic acid
was more likely owing to the chelating property of tannic acid
(Maqsood & Benjakul, 2010a). Tannic acid is able to chelate iron
particularly in free form (Maqsood & Benjakul, 2010a). As a conse-
quence, non-haem iron content in tannic acid treated samples was
lower than those without tannic acid treatment (P < 0.05). During
storage, non-haem iron was released due to the deterioration by
microbial spoilage (Maqsood & Benjakul, 2010a). Those free irons
might accelerate the oxidation process in the ground beef. After
15 days of storage, samples stored under MAP with high oxygen
concentration without tannic acid treatment M2(T) tended to
contain higher content of non-haem iron than other samples.
Under high oxygen atmosphere, the spoilage microorganisms grew
rapidly and destructed haem, resulting in more release of non-
haem iron. Among the transition metals, iron particularly ferrous
form is known as the most important pro-oxidant due to its high
reactivity (Love, 1983). The ferrous state of iron accelerates lipid
oxidation by breaking down hydrogen peroxide and lipid peroxides
to reactive free radicals via the Fenton reaction (Fe2+ + H2O2 ?
Fe3+ + OH + OH) (Maqsood & Benjakul, 2010a, 2010b). Therefore,
the application of tannic acid having metal chelating ability in con-
junction with MAP could lower the lipid oxidation mediated by
transition metal ions in ground beef.
3.1.3. Changes in oxymyoglobin and metmyoglobin percentage
Changes in oxymyoglobin and metmyoglobin percentages in
ground beef treated without and with tannic acid and stored in
air and under MAP with different gas compositions during refriger-
ated storage are presented in Fig. 3a and b, respectively. Oxymyo-
globin in all samples decreased gradually throughout the storage of
15 days. The decreases were more pronounced within the rst
3 days of storage. The higher decrease in oxymyoglobin was
noticeable in samples stored in the air and under MAP with low
1286 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290
A1(+T)
80
70
Oxymyoglobin (%)
60
50
40
30
20
10
0
0 3
A1(+T)
100
Metmyoglobin (%)
80
60
40
20
0
0 3
Fig. 3. Changes in oxymyoglobin (a) and metmyoglobin (b) percentages in ground beef treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/
70%N2 or 80%O2/20%CO2) during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).
oxygen concentration, compared with those kept under MAP with
high oxygen concentration. The results suggested that the atmo-
sphere rich in oxygen could retard the oxidation of oxymyoglobin
during the extended storage. Tang et al. (2006) reported the
decrease in oxymyoglobin in minced beef patties held in air and
under MAP (80%O2/20%CO2) with increasing storage time. MAP
with high oxygen was effective in maintaining the myoglobin in
its oxygenated form. The higher oxymyoglobin in the ground beef
stored under MAP with high oxygen was related with the bright
red colour of ground beef during the refrigerated storage. High
oxygen atmosphere (6080%) maintains the bright red colour of
meat (Okayama et al., 1995), in which myoglobin was present in
its oxygenated form (OGrady et al., 2000).
Metmyoglobin formation of ground beef increased with
increasing storage time (P < 0.05). The increase in metmyoglobin
was coincidental with the decrease in oxymyoglobin (Fig. 3a). Tang
et al. (2006) also reported the increase in metmyoglobin in minced
beef patties held in air and under MAP (80%O2 and 20%CO2) as the
time of storage increased. The ground beef stored under MAP with
high oxygen concentration showed the lower metmyoglobin for-
mation, compared with those kept in air and under MAP with
low oxygen concentration (P < 0.05). Ordonez and Ledward
(1977) found that increasing oxygen concentration caused a signif-
icant decrease in the metmyoglobin (MetMb) formation in pork
meat and less than 30% of MetMb of the total surface pigment con-
centration was obtained after 15 days of storage in 80%O2/20%CO2.
The higher metmyoglobin formation in the ground beef kept in air
and under MAP with low oxygen concentration correlated well
with lower oxymyoglobin percentage in those samples. Metmyo-
globin formation is related with browning in the red meats (Bou
et al., 2008). At day 15, the samples stored under MAP with low
oxygen showed the highest metmyoglobin formation. In general,
A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)
6 9 12 15
Storage time (days)
(a)
A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)
6 9 12 15
Storage time (days)
(b)
the treatment of tannic acid had no effect on oxymyoglobin and
metmyoglobin formation. The ground beef packed under MAP with
high oxygen concentration (80%) had the lower metmyoglobin and
higher oxymyoglobin throughout the refrigerated storage of
15 days. With the treatment using tannic acid, the samples kept
under MAP with high oxygen exhibited the lower metmyoglobin
in comparison with non-treated counterparts after 15 days of
refrigerated storage (P < 0.05). This was coincidental with the
higher oxymyoglobin in the former (P < 0.05).
3.1.4. Changes in colour
Changes in colour (L, a and b) of the ground beef treated with-
out and with tannic acid and stored in air and under MAP during
the refrigerated storage are depicted in Fig. 4. The a (redness) va-
lue of all the samples decreased gradually during the storage
(P < 0.05), however the rate of decrease varied with the treatments.
The decrease in a value was in agreement with the oxidation of
myoglobin and accumulation of metmyoglobin (Mancini & Hunt,
2005). Decreasing a values with increasing storage time were re-
ported in beef burgers during frozen storage (Georgantelis, Blekas,
Katikou, Ambrosiadis, & Fletouris, 2007). When meat losses its
ability to reduce metmyoglobin to oxymyoglobin, the brown col-
our of metmyoglobin begins to appear on the surface of beef
steaks, and a values begins to decrease (Friedrich et al., 2008).
The samples kept under MAP with high oxygen concentration
showed the highest a value, regardless of tannic acid treatment.
Those packed in air and under MAP with low oxygen concentration
displayed the lower a value (P < 0.05). This reconrmed the essen-
tial role of oxygen in oxygenation of myoglobin in beef. The refrig-
erated beef steaks stored under MAP with high oxygen
concentration (5080%) had the higher a values when compared
with those stored under MAP with low oxygen concentration
 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290 1287

A1(+T) A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)

50

45

L*-value
40

35

30
0 3 6 9 12 15
Storage time (days)

(a)
A1(+T) A1 (-T) M1(+T) M1(-T) M2(+T) M2(-T)
25

20
a*-value

15

10

5
0 3 6 9 12 15
Storage time (days)

(b)
A1(+T) A1(-T) M1(+T) M1(-T) M2(+T) M2(-T)
25
20
b*-value

15
10
5
0
0 3 6 9 12 15
Storage time (days)

(c)
Fig. 4. Changes in L (a), a (b) and b (c) values of ground beef treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/70%N2 or 80%O2/20%CO2)
during 15 days of refrigerated storage. Bars represent the standard deviation (n = 3).

(020%) (Zakrys, Hogan, OSullivan, Allen, & Kerry, 2008). The higher
redness (a values) in M2(T) and M2(+T) samples was associated
with the higher percentage of oxymyoglobin in these samples
(Fig. 3a). Therefore, high-oxygen atmospheres (80%O2) promote
the oxygenation of pigments, thereby prolonging the time before
metmyoglobin is visible on the muscle surface (Jayasingh et al.,
2002). The lower redness in the sample stored in air and under
low oxygen MAP was attributed to the rapid oxidation of oxymyo-
globin into metmyoglobin, resulting in the increased browning in
those samples.
No changes in L value were found in all samples during the
refrigerated storage of 15 days (P > 0.05). Friedrich et al. (2008) re-
ported slight changes in the L values of the minced beef in the
oxygen rich atmosphere during chilled storage. The samples stored
under MAP with low oxygen concentration showed the higher L
values than those stored under MAP with high oxygen concentra-
tion, especially after 9 days of storage (P < 0.05). The b-value indi-
cating yellowness of all samples except A1(+T) increased slightly
within the rst 3 days of storage (P < 0.05). Thereafter the b-values
remained more or less constant (P > 0.05). Among all samples,
M2(T) showed the higher b-values, suggesting the higher yel-
lowness of the sample. Under the high oxygen atmosphere in the
absence of tannic acid, lipid oxidation could take place (Fig. 1b). Li-
pid oxidation products, especially carbonyl compounds, could un-
dergo Maillard reaction in the presence of amino groups in beef.
The loss in redness (a) and the increase in yellowness (b) corre-
lated well with the formation of metmyoglobin along with the de-
crease in oxymyoglobin in the beef (Fig. 3). The treatment of
ground beef with tannic acid could lower the b-values of samples
kept under MAP with high oxygen. This suggested the ability of
tannic acid in inhibiting lipid oxidation, where yellowness could
be developed in the beef. Thus, the high oxygen MAP could main-
tain the redness up to 12 days of refrigerated storage, irrespective
of tannic acid treatment.
3.1.5. Changes in protein patterns
Protein patterns of fresh ground beef (day 0) and ground beef
treated without and with tannic acid stored in air and under
MAP for 15 days are shown in Fig. 5. All samples contained myosin
heavy chain (MHC) and actin as the major proteins. Additionally,
all samples contained tropomyosin, troponin-T and troponin-C.
MHC and actin underwent degradation after 15 days of storage at
4 C at different degrees, depending upon the treatments. Under
MAP with high oxygen concentration, band intensity of MHC of
M2(T) sample decreased by 29.76%, whereas that of M2(+T) sam-
ple decreased by 25.93% after 15 days of storage. The results sug-
gested that tannic acid could lower the degradation caused by
aerobic microorganisms. On the other hand, samples stored under
MAP with low oxygen concentration had the lower degradation,
particularly with tannic acid treatment. For M1(+T) samples,
MHC was degraded by 8.01%. This was mainly due to the synergis-
tic effect of low oxygen MAP and tannic acid on retardation of the
growth of microorganisms with proteolytic activity. Apart from
MHC, actin was also degraded after 15 days of storage when com-
1288 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290

F A1 M1 M2 observed (P < 0.05). Under the same packaging atmosphere, the

kDa
200
116
97
66
55
45
36
29
24
20
M +T -T +T -T +T -T
Fig. 5. SDSPAGE patterns of muscle protein from fresh ground beef (F) and sample
treated without and with tannic acid stored in air (A1) and under MAP with 10%O2/
20%CO2/70%N2 (M1) or 80%O2/20%CO2 (M2) after 15 days of refrigerated storage. M:
marker; +T: with tannic acid treatment; T: without tannic acid treatment.
pared with that found in the fresh sample. Actin was degraded by
25.9% in samples stored under MAP with high oxygen concentra-
tion without tannic acid treatment M2(T) and by 1.85% for the
samples kept under MAP with low oxygen concentration and trea-
ted with tannic acid, M1(+T). The higher degradation of MHC and
actin in sample kept under MAP with high oxygen without tannic
acid treatment, M2(T) might be due to the higher degree of pro-
teolysis associated with the pronounced microbial growth. More-
over, protein oxidation (Park, Xiong, & Alderton, 2007) and
endogenous and microbial proteinases such as cathepsin and cal-
pain can also result in protein degradation (Masniyom, Benjakul,
& Visessanguan, 2004). Lund et al. (2007) reported that MHC was
found to be slightly less intense in the high oxygen stored beef
due to cross linking caused by protein oxidation. Troponin and
tropomyosin were also degraded to a higher extent when the sam-
ples were stored under MAP with high oxygen concentration. Thus,
the tannic acid could prevent protein degradation of refrigerated
ground beef to some degree and packaging atmosphere played
more important role in protein degradation in beef during the
refrigerated storage.
3.2. Effect of tannic acid in combination without and with MAP on the
microbiological changes of ground beef during refrigerated storage
Psychrophilic bacterial count (PBC) of ground beef treated with-
out and with tannic acid and stored in air and under MAP at the
day 0 and 15 of refrigerated storage is shown in Fig. 6. PBC of all
samples increased from an initial value of 2.69  101 cfu/g to
7.69.96  108 cfu/g at the end of storage (day 15) (P < 0.05). When
MAP with low oxygen concentration was used, the lower PBC was
MHC (200 kDa)
Actin (44 kDa),
Tropomyosin (38 kDa)
Troponin-T (32 kDa)
Troponin-C (30 kDa)
and Kouno (2004) reported the antibacterial activity of hydrolysa-
Whittier (2001) stated that panelist description of brown beef is
samples treated with tannic acid had the lower PBC, compared
with non-treated counterparts (P < 0.05). PBC of ground beef with-
out tannic acid treatment and stored under MAP with high oxygen
concentration was similar to the sample kept in air [A1(T)]
(P > 0.05). Viana, Gomide, and Vanetti (2005) found the pro-
nounced growth of psychrophilic bacteria Pseudomonas in beef
(loin) samples stored under MAP (100%O2) than found in samples
kept in air. The result suggested that the tannic acid was effective
in retarding the microbial growth even in MAP with high oxygen.
Tannic acid could act as antimicrobial agent (Zaidi-Yahiaoui, Zaidi,
& Ait Bessai, 2008). Grape seed extract and pine bark extract
containing tannin at a level of 1% rapidly reduced the numbers of
Escherichia coli O157:H7 in cooked beef within the rst 3 days of
storage at 4 C (Ahn, Grun, & Mustapha, 2007). Taguri, Tanaka,
ble tannins against Salmonella aureus and Staphylococcus. Thus, the
use of tannic acid in combination with high and low oxygen MAP
exhibited the inhibitory effect on the growth of psychrophilic bac-
teria which were dominant at refrigerated storage temperature.
3.3. Effect of tannic acid in combination without and with MAP on the
sensorial changes of ground beef during refrigerated storage
Scores of colour and odour likeness of ground beef treated with-
out and with tannic acid and stored in air and under MAP are
shown in Table. 1. The likeness scores for both attributes continu-
ously decreased with increasing storage time (P < 0.05). During the
storage, the highest colour likeness scores were obtained in ground
beef stored under high oxygen MAP, while the sample stored in air
and under low oxygen MAP had the lower scores in descending or-
der. This was mainly due to the higher percentage of oxymyoglobin
present in the ground beef stored under high oxygen MAP (Fig. 3a).
Under the same packaging conditions, tannic acid treatment had
no impact on colour likeness of ground beef. However, a non-sig-
nicantly lower score of colour likeness was found in the samples
treated with tannic acid. This can be attribute to the fact that oxi-
dation of oxymyoglobin might take place. Tannic acid might have
the interaction with the globin protein chain, thereby altering
the tertiary structure and potentially opening the haem cleft. This
change could destabilize the haem, resulting in enhanced oxidation
of oxymyoglobin along with the formation of metmyoglobin
(Hayes et al., 2009). The samples stored in air and under low oxy-
gen MAP received the lowest colour scores and were rejected by
the panelists from the day 9 onwards of refrigerated storage. These
samples turned to brownish rapidly, which correlated well with
the high metmyoglobin formation (Fig. 3b) and lowered redness
(a) values (Fig. 4a) in those samples. Carpenter, Cornforth, and
associated with a decrease in redness (a) values. Similar nding

A1 (+T) A1 (-T) M1(+T) M1 (-T) M2 (+T) M2 (-T)

Psychrophilic Bacterial

12 a
a
10 c b c
d
(CFU/g)

8
Count

6
4 a a a a a a

2
0
0 15
Storage time (days)

Fig. 6. Changes in psychrophilic bacterial count (PBC) in fresh ground beef and sample treated without and with tannic acid stored in air and under MAP (10%O2/20%CO2/
70%N2 or 80%O2/20%CO2) after 15 days of refrigerated storage. Bars represent the standard deviation (n = 3). Different letters on the bar within the same storage time denote
the signicant differences (P < 0.05).
 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290 1289

Table 1
Likeness scores of ground beef without and with tannic acid treatment and stored in air and under MAP (10%O2/20%CO2/70%N2 or 80%O2/20%CO2) during 15 days of refrigerated
storage.

Attributes Samples Day 0 Day 3 Day 9 Day 15

Colour A1(+T) 9.20 0.34Aa 6.40 0.44Bc 5.10 0.48Ce 3.40 0.41Dd
A1(T) 9.40 0.45Aa 6.70 0.36Bc 5.60 0.37Ce 3.60 0.64Dd
M1(+T) 9.30 0.49Aa 6.20 0.51Bc 4.20 0.28Ce 3.00 0.37Dd
M1(T) 9.10 0.29Aa 6.30 0.48Bc 4.60 0.46Ce 3.20 0.30Dd
M2(+T) 9.40 0.48Aa 8.40 0.44Bb 6.90 0.42Cad 5.30 0.38Dab
M2(T) 9.70 0.42Aa 8.50 0.39Bb 7.10 0.43Ccd 5.80 0.31Da
Odour A1(+T) 10.0 0.38Aa 9.10 0.41Ba 7.70 0.45Cc 5.10 0.33Db
A1(T) 9.90 0.49Aa 9.30 0.48Ba 7.20 0.39Cbc 4.50 0.29Dc
M1(+T) 9.80 0.57Aa 8.80 0.33Bab 6.90 0.42Cab 5.00 0.28Dac
M1(T) 9.90 0.22Aa 8.70 0.30Bab 6.60 0.38Ca 4.10 0.39Dc
M2(+T) 9.80 0.38Aa 8.90 0.39Bab 6.80 0.41Ca 5.30 0.33Dab
M2(T) 9.80 0.43Aa 9.00 0.37Ba 6.50 0.39Ca 4.60 0.39Dc

Values with the same capital superscript in the same row do not differ signicantly (P > 0.05). Values with same superscript in the same column within same attribute do not
differ signicantly (P > 0.05).

was also reported by Jayasingh et al. (2002) who found that ground
beef packed in high oxygen MAP maintained a bright red colour for
10 days. John et al. (2005) reported that beef steaks packed under
high oxygen atmosphere had a desirable red colour on day 7 of
storage, but some browning was evident by day 14 and steaks were
completely brown and unappealing by day 21.
The likeness scores for odour of all samples also decreased with
the increasing storage time (P < 0.05). The samples without tannic
acid treatment, regardless of the packaging conditions, received
the lower odour scores, comparable with those treated with tannic
acid (P < 0.05). This was attributed to higher TBARS formation
(Fig. 1b) and higher PBC (Fig. 6) in those samples without tannic
acid treatment. The panelists detected off-odour mainly in the
sample stored in high oxygen MAP, which was mainly due to the
oxidative rancidity. St. Angelo, Crippen, Dupuy, and James (1990)
reported that lipid oxidation causes the deterioration of avour
in beef stored under atmospheres enriched in O2 and this deterio-
ration can be closely related to TBARS. The odour scores were
higher for all ground beef samples treated with tannic acid under
all packaging conditions (P < 0.05). Tannic acid could lower the
oxidation of lipids in muscle foods (Maqsood & Benjakul, 2010a)
and retard the microbial growth (Zaidi-Yahiaoui et al., 2008). Thus,
the development of rancidity and off-odour in samples without
tannic acid treatment was in accordance with the increased PBC
(Fig. 6) and increased TBARS values (Fig. 1b).
4. Conclusion
Tannic acid at a level of 200 ppm in combination with high oxy-
gen concentration MAP (80%O2 and 20%CO2) could be effective in
maintaining the bright red colour and retarding the lipid oxidation
and microbial growth in ground beef up to 15 days of refrigerated
storage. Nevertheless, the protein degradation still proceeded to
some degree. Therefore, tannic acid can be a natural additive with
multiple preservative functions in meat and meat products.
Acknowledgements
The authors would like to express their sincere thanks to Grad-
uate School of Prince of Songkla University and The Commission of
Higher education, Ministry of Education, Thailand for the nancial
support.
References
Ahn, J., Grun, I. U., & Fernando, L. N. (2002). Antioxidant properties of natural plant
extracts containing polyphenolic compounds in cooked ground beef. Journal of
Food Science, 67, 13641369.
Ahn, J., Grun, I. U., & Mustapha, A. (2007). Effects of plant extracts on microbial
growth, colour change, and lipid oxidation in cooked beef. Food Microbiology, 24,
714.
Benjakul, S., & Bauer, F. (2001). Biochemical and physicochemical changes in catsh
(Silurus glanis L) muscle as inuenced by different freezethaw cycles. Food
Chemistry, 72, 207217.
Boselli, E., Caboni, M. F., Rodriguez-Estrada, M. T., Toschi, T. G., Daniel, M., & Lercker,
G. (2005). Photoxidation of cholesterol and lipids of turkey meat during storage
under commercial retail conditions. Food Chemistry, 91, 705713.
Bou, R., Guardiola, F., Codony, R., Faustman, C., Elias, R. J., & Decker, E. A. (2008).
Effect of heating oxymyoglobin and metmyoglobin on the oxidation of muscle
microsomes. Journal of Agricultural and Food Chemistry, 56, 96129620.
Carlez, A., Veciana-Nogues, T., & Cheftel, J. C. (1995). Changes in colour and
myoglobin of minced beef meat due to high pressure processing. Lebensm.-Wiss.
u. Technology, 28, 528538.
Carpenter, C. E., Cornforth, D. P., & Whittier, D. (2001). Consumer preferences for
beef colour and packaging did not affect eating satisfaction. Meat Science, 57,
359363.
Cousin, M. A., Jay, J. M., & Vasavada, P. C. (1992). Psychrotrophic microorganism. In
C. Vanderzand & D. F. Splittstoesser (Eds.), Compendium of methods for the
microbiological examination of foods (pp. 153168). Washington, DC: American
Public Health Association.
Decker, E. A., & Hultin, H. O. (1990). Factors inuencing catalysis of lipid oxidation
by the soluble fraction of mackerel muscle. Journal of Food Science, 55, 947950.
Friedrich, L., Siro, I., Dalmadi, I., Horvath, K., Agoston, R., & Balla, Cs. (2008).
Inuence of various preservatives on the quality of minced beef under modied
atmosphere at chilled storage. Meat Science, 79, 332343.
Georgantelis, D., Blekas, G., Katikou, P., Ambrosiadis, I., & Fletouris, D. J. (2007).
Effect of rosemary extract, chitosan and a-tocopherol on lipid oxidation and
colour stability during frozen storage of beef burgers. Meat Science, 75, 266274.
Gomez-Basauri, J. V., & Regenstein, J. M. (1992). Vacuum packaging, ascorbic acid
and frozen storage effects on heme and non-heme iron content of mackerel.
Journal of Food Science, 57, 13371339.
Hayes, J. E., Stepanyan, V., Allen, P., OGrady, M. N., OBrien, N. M., & Kerry, J. P.
(2009). The effect of lutein, sesamol, ellagic acid and olive leaf extract on lipid
oxidation and oxymyoglobin oxidation in bovine and porcine muscle model
systems. Meat Science, 83, 201208.
Jayasingh, P., Cornforth, D. P., Brennand, C. P., Carpenter, C. E., & Whittier, D. R.
(2002). Sensory evaluation of ground beef stored in high-oxygen modied
atmosphere packaging. Journal of Food Science, 67, 34933496.
John, L., Cornforth, D., Charles, E., Sorheim, O., Pettee, B. C., & Whittier, D. R. (2005).
Colour and thiobarbituric acid values of cooked top sirloin steaks packaged in
modied atmospheres of 80% oxygen or 0.4% carbon monoxide, or vacuum.
Meat Science, 69, 441449.
Krywicki, K. (1982). The determination of haem pigment from fresh meat. Meat
Science, 7, 2936.
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of 17
bacteriophage T4. Nature, 227, 680685.
Love, J. D. (1983). The role of heme iron in the oxidation of lipids in red meats. Food
Technology, 37, 117120.
Luciano, G., Monahan, F. J., Vasta, V., Pennisi, P., Bella, M., & Priolo, A. (2009). Lipid
and colour stability of meat from lambs fed fresh herbage or concentrate. Meat
Science, 82, 193199.
Lund, M. N., Hviid, M. S., & Skibsted, L. H. (2007). The combined effect of
antioxidants and modied atmosphere packaging on protein and lipid oxidation
in beef patties during chill storage. Meat Science, 76, 226233.
Mailgaad, M., Civille, G. V., & Carr, B. T. (1999). Sensory evaluation techniques. Boca
Raton, FL: CRS Press.
Mancini, R. A., & Hunt, M. C. (2005). Current research in meat colour. Meat Science,
71, 100121.
Maqsood, S., & Benjakul, S. (2010a). Comparative studies of four different phenolic
compounds on in vitro antioxidative activity and the preventive effect on lipid
oxidation of sh oil emulsion and sh mince. Food Chemistry, 119, 123132.
1290 S. Maqsood, S. Benjakul / Food Control 21 (2010) 12821290
Maqsood, S., & Benjakul, S. (2010b). Synergistic effect of tannic acid and modied
atmospheric packaging on the prevention of lipid oxidation and quality losses
of refrigerated striped catsh slices. Food Chemistry, 121, 2938.
Masniyom, P., Benjakul, S., & Visessanguan, W. (2004). ATPase activity, surface
hydrophobicity, sulfhydryl content and protein degradation in refrigerated
seabass muscle in modied atmosphere packaging. Journal of Food Biochemistry,
28, 4360.
Morrissey, P. A., Sheehy, P. J. A., Galvin, K., Kerry, J. P., & Buckley, D. J. (1998). Lipid
stability in meat and meat products. Meat Science, 49, 7386.
OGrady, M. N., Monahan, F. J., Burke, R. M., & Allen, P. (2000). The effect of oxygen
level and exogenous a-tocopherol on the oxidative stability of minced beef in
modied atmosphere packs. Meat Science, 55, 3945.
Okayama, T., Muguruma, M., Murakami, S., & Yamada, H. (1995). Effect of modied
atmosphere packaging systems on pH value, microbial growth, metmyoglobin
formation and lipid oxidation of thin sliced beef. Journal of the Japanese Society
for Food Science and Technology [Nippon Shokuhin Kagaku Kogaku Kaishi], 42,
498504.
Ordonez, J. A., & Ledward, D. A. (1977). Lipid and myoglobin oxidation in pork
stored in oxygen- and carbon dioxide-enriched atmospheres. Meat Science, 1,
4148.
Park, D., Xiong, Y. L., & Alderton, A. L. (2007). Concentration effects of hydroxyl
radical oxidizing systems on biochemical properties of porcine muscle
myobrillar protein. Food Chemistry, 3, 12391246.
Schricker, B. R., Miller, D. D., & Stouffer, J. R. (1982). Measurement and content of
non-heme and total iron in muscle. Journal of Food Science, 47, 740743.
St. Angelo, A. J., Crippen, K. L., Dupuy, H. P., & James Jr., (1990). Chemical and sensory
studies of antioxidant treated beef. Journal of Food Science, 55, 15011505.
Steel, R. G. D., & Torrie, J. H. (1980). Principles and procedures of statistics. New York:
McGraw-Hill Book Co.. pp. 106107.
Taguri, T., Tanaka, T., & Kouno, I. (2004). Antimicrobial activity of 10 different plant
polyphenols against bacteria causing food-borne disease. Biological and
Pharmaceutical Bulletin, 27, 19651969.
Tang, S. Z., Ou, S. Y., Huang, X. S., Li, W., Kerry, J. P., & Buckley, D. J. (2006). Effects of
added tea catechins on colour stability and lipid oxidation in minced beef
patties held under aerobic and modied atmospheric packaging conditions.
Journal of Food Engineering, 77, 248253.
U.S. Code of Federal Regulations (2006). Tannic acid. 21 CFR 184.1097, pp. 492493.
Viana, E. S., Gomide, L. A. M., & Vanetti, M. C. D. (2005). Effect of modied
atmospheres on microbiological, colour and sensory properties of refrigerated
pork. Meat Science, 71, 696705.
Zaidi-Yahiaoui, R., Zaidi, F., & Ait Bessai, A. (2008). Inuence of gallic and tannic acid
on enzymatic activity and growth of Pectobacterium chrysanthemi (Dickeya
chrysanthemi bv. chrysanthemi). African Journal of Biotechnology, 7, 482486.
Zakrys, P. I., Hogan, S. A., OSullivan, M. G., Allen, P., & Kerry, J. P. (2008). Effects of
oxygen concentration on the sensory evaluation and quality indicators of beef
muscle packed under modied atmosphere. Meat Science, 79, 648655.