MEAT

SCIENCE
Meat Science 75 (2007) 256–264
www.elsevier.com/locate/meatsci

Effect of rosemary extract, chitosan and a-tocopherol on lipid

oxidation and colour stability during frozen storage of beef burgers
Dimitrios Georgantelis a,*, Georgios Blekas b, Panagiota Katikou a,
Ioannis Ambrosiadis a, Dimitrios J. Fletouris a
a
Department of Hygiene and Technology of Animal Origin Products, School of Veterinary Medicine,
Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
b
Laboratory of Food Chemistry and Technology, School of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

Received 2 May 2006; received in revised form 10 July 2006; accepted 13 July 2006

Abstract

The effect of rosemary extract, chitosan and a-tocopherol, added individually or in combination, on lipid oxidation and colour sta-
bility of frozen (18 C) beef burgers stored for 180 days was investigated. The burgers’ lipid oxidation and appearance were evaluated
through measurement of primary (conjugated dienes and peroxide value) and secondary (malondialdehyde) oxidation products, together
with visual assessment and instrumental measurement of colour. Chitosan alone and in combination with either rosemary or a-tocoph-
erol had the best antioxidative effect (P 6 0.05) compared to individual use of rosemary or a-tocopherol, while the best results were
obtained with the combination of chitosan and rosemary. The differences of antioxidative effects, however, between individual use of
rosemary or a-tocopherol as compared to the controls were also significant (P 6 0.05). Chitosan added individually or in combination
with either rosemary or a-tocopherol had also a noteworthy effect on the burgers’ appearance as it contributed to red colour retention for
a much longer period (P 6 0.05) compared all other treatments and the controls. In conclusion, the best antioxidative effects were
obtained with the combination of chitosan and rosemary extract.
 2006 Elsevier Ltd. All rights reserved.

Keywords: Beef burgers; Chitosan; Colour; Lipid oxidation; Natural antioxidants; Rosemary; a-tocopherol

1. Introduction tion results in the formation of pro-oxidants capable of

Lipid oxidation, leading to rancidity, is one of the major
reasons of meat products’ quality deterioration, mainly
because of their increased fat content, the highly commi-
nuted nature of the raw materials, as well as the possibility
of long-term frozen storage of the latter before use. This
irreversible change contributes to the development of unac-
ceptable organoleptic characteristics and is more important
in frozen preserved meat products, where microbiological
spoilage is not common. Oxidative processes are also asso-
ciated with discolouration of meat products, as lipid oxida-
*
Corresponding author. Present address: 9 Triantafyllopoulou St.,
54352 Thessaloniki, Greece. Tel./fax: +30 2310 938627.
E-mail address: dimitrisgeorgantelis@yahoo.gr (D. Georgantelis).
reacting with oxymyoglobin, which lead to metmyoglobin
formation (Frankel, 1998). Appearance of meat products
is one of the major determinants of their appeal to consum-
ers and, consequently, sales of the product (Clydesdale,
1998). Williams et al. (1992) have investigated the econom-
ical importance of colour deterioration of meat and meat
products. Their data indicated that this problem is associ-
ated with a sales reduction of 5.4% for fresh meat and
3.7% for meat products, whereas its prevention could lead
to a saving of profit.
In order to protect lipids and avoid deterioration of
appearance, meat product manufacturers in the past few
decades have used several food additives with antioxidative
properties. The increasing consumer awareness and health-
consciousness, however, results in pressure to avoid the use

0309-1740/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2006.07.018
 D. Georgantelis et al. / Meat Science 75 (2007) 256–264
of synthetic additives, which necessitates the use of natural
additives or alternative methods to extend shelf life and/or
improve safety. One such solution could be the use of nat-
ural antioxidants.
Tocopherols are effective natural antioxidants for lipid
containing foods. a-tocopherol behaves like a chain-break-
ing electron donor antioxidant by competing with the
substrate for the chain-carrying peroxyl radicals. More-
over, a-tocopherol has also been associated with retarding
the decomposition of hydroperoxides (Frankel, 1998).
Although much research has focused on supplementation
of animal feed with a-tocopherol for improving myoglobin
and lipid stability of beef (Lanari, Cassens, Schaefer, &
Scheller, 1994), pork (Buckley, Morrissey, & Gray, 1995),
turkey (Mercier, Gatellier, Viau, Remignon, & Renerre,
1998) and lamb (Guidera, Kerry, Buckley, Lynch, & Mor-
rissey, 1997) meat, few studies have been conducted as
regards to the in vitro treatment of ground beef meat with
a-tocopherol.
Rosemary (Rosmarinus officinalis L.) extracts have also
exhibited potent antioxidant activity and are widely used
in the food industry. The antioxidant activity of rosemary
extracts has been associated with the presence of several
phenolic diterpenes such as carnosic acid, carnosol, rosma-
nol, rosmariquinone and rosmaridiphenol, which break
free radical chain reactions by hydrogen atom donation
(Aruoma, Halliwell, Aeschbach, & Lóliger, 1992; Basaga,
Tekkaya, & Acitel, 1997). A number of researchers have
reported the effectiveness of rosemary extracts for achiev-
ing higher sensory scores and retarding lipid oxidation in
various foods: Stoick, Gray, Booren, and Buckley (1991)
used 500–1000 ppm in beef steaks; Shahidi and Wanasund-
ara (1992) recommended concentrations ranging between
200 and 1000 ppm in various foods, while Sebranek, Sew-
alt, Robbins, and Houser (2005) reported that the addition
of 1000 ppm of rosemary extract was as effective as BHA/
BHT in maintaining low TBARS values of precooked-fro-
zen sausage. In addition to inhibition of lipid oxidation,
Formanek, Lynch, Galvin, Farkas, and Kerry (2003) found
that colour changes during storage were inhibited by the
addition of rosemary extract in irradiated ground beef.
Chitosan, which is the deacetylated form of chitin, has
been identified as a versatile biopolymer for a broad range
of food applications (Shahidi, Arachchi, & Jeon, 1999).
Effectiveness of chitosan treatment on oxidative stability
of minced beef was studied by Darmadji and Izumimoto
(1994) who observed that the addition of chitosan at 1%
resulted in a 70% decrease in the 2-thiobarbituric acid
(TBA) values of meat after 3 days storage at 4 C. The
mechanism by which this inhibition takes place is believed
to be related to the chelation of free iron, which is released
from the hemoproteins of meat during heat processing or
storage. This would in turn inhibit the catalytic activity
of iron ions (Kamil, Jeon, & Shahidi, 2002).
Thus several studies have demonstrated the beneficial
effects of these natural antioxidants on lipid oxidation
and colour preservation. Due to the interaction of these
257
compounds with their environment, however, the investi-
gation of their activity in a range of food systems is still
needed for successful application in meat products. The
objective of the present research was to determine the
effect of rosemary extract, chitosan and a-tocopherol,
applied individually or in combination, on lipid oxida-
tion and colour stability during frozen storage of beef
burgers.
2. Materials and methods
2.1. Natural antioxidants and chemicals
The a-tocopherol oil preparation (670 mg/g; 1000 IU/g;
storage at 20 C) was obtained from Sigma (Sigma–
Aldrich Inc., St. Louis, MO, USA). Rosemary extract
(Stabiloton OS) containing 30 ± 3% phenolic diterpenes
(carnosic acid, carnosol, rosmanol and rosmarinic acid),
was purchased from RAPS GmbH & Co. (Kulmbach, Ger-
many). Food grade chitosan (MW: 4.9 · 105, degree of
deacetylation: 88.2%, viscosity: 75 cps) was procured from
Dalian Xindie Chitin Co. Ltd. (Dalian, Liaoning, China).
All other chemicals used were of analytical grade or the
highest grade available and were obtained either from
Sigma–Aldrich or Merck (Darmstadt, Germany).
2.2. Preparation of burgers
Beef brisket and flank (carcass quality grade R3) were
obtained 48 h postmortem, their fat contents were deter-
mined and they were then frozen at 20 C until process-
ing. After thawing, they were cut in small cubes and
comminuted separately at 2 C through a 6 mm steel
plate (Maskinfabriken Strommen a/s, Denmark). The
raw materials were then thoroughly mixed at an appropri-
ate ratio to achieve a fat content of approximately 15% in
the mixture and were comminuted again through a 4 mm
steel plate. The mixture was divided into six equal parts
to prepare the experimental treatments.
Each of these parts was transferred to a commercial
mixer (Kenwood chef-excel) where they were mixed for
8 min with salt (1.5% w/w), corn oil (0.15% w/w) and the
appropriate antioxidants, except for one part, which served
as the control (Table 1). Rosemary extract was added at a
concentration of 200 mg/kg, within the range of 150–
300 mg/kg suggested for hamburgers by the manufacturer.
Chitosan was used at the level of 10 mg/kg, which showed
the best antioxidative effect in the study of Darmadji and
Izumimoto (1994). The selected addition level for
a-tocopherol was 60 mg/kg, as Wong, Hashimoto, and Shi-
bamoto (1995) in meat model systems have reported simi-
lar antioxidative effects for a-tocopherol between 50 and
100 mg/kg, while other studies indicated the presence of a
pro-oxidative effect at a-tocopherol concentrations greater
than 100 mg/kg (Lampi & Piironen, 1998; Verma & Sahoo,
2000). Corn oil was used as the carrier of the a-tocopherol
preparation in order to achieve better distribution in the
258 D. Georgantelis et al. / Meat Science 75 (2007) 256–264
Table 1
Addition of antioxidants for the preparation of experimental treatments of
burgers
Antioxidant Treatment
R+C ROS T+C CHI
Rosemary extract + + – –
(200 mg/kg)
Chitosan (10 g/kg) + – + +
a-tocopherol (60 mg/kg) – – + –
meat blend and was also added to the remaining treatments
for uniformity.
The meat blend for each treatment was then portioned
into acrylic glass templates of 10 cm diameter and 8 mm
height. Throughout the procedure, the temperature of the
meat blend remained close to 0 C. The burgers were then
packed in air-permeable polypropylene bags, frozen to
70 C until the core temperature reached 18 C and
they were then stored at 18 C for 180 days. The whole
procedure was standardised to keep the time between the
processing steps equal for all burgers. All experiments were
repeated three times, in order to remove any effects deriv-
ing from the initial quality of the raw material.
2.3. Sampling
For all tested parameters, determinations were per-
formed on days 0, 30, 60, 90, 120, 150 and 180 of frozen
storage on two separate samples in duplicate. On day 0,
chemical analyses for the assessment of lipid oxidation
were performed only for the control samples, whereas
instrumental measurements for colour were conducted for
all treatments.
2.4. Proximate analysis and fatty acid profile of the meat
mixture
Chemical composition (moisture, protein, fat and ash)
and fatty acid profile were determined only in the initial
meat mixture used for the preparation of the experimental
burgers, before the addition of corn oil. Moisture content
was determined by drying the homogenised sample in an
oven at 105 C until a constant weight was obtained. Crude
protein content was calculated by converting the nitrogen
content determined by Kjeldahl’s method (6.25 · N). Fat
was determined by the method described by the AOAC
(2003) using a Soxhlet automated system. Ash content
was determined by dry-ashing in a furnace at 525 C for
24 h (AOAC, 2003). Fatty acids were extracted and con-
verted to methylesters using the method of Folch, Lees,
and Sloane-Stanley (1957). They were then analysed in a
Hewlett Packard 5890 gas chromatographer (Palo Alto,
CA, USA) according to the method described in Annex
XA of the EU Regulation 2568/91. Results were reported
as per cent content (w/w) of individual fatty acid to the
sum of the total fatty acid content. All analyses were per-
formed in duplicate.
2.5. Measurement of lipid oxidation
Lipid oxidation was assessed through determination of
primary (conjugated dienes and hydroperoxides) and sec-
TOC CON ondary (malondialdehyde) oxidation products formed dur-
– – ing frozen storage. Conjugated dienes and hydroperoxides
were determined in the lipids extracted from the burgers on
– – each sampling day, according to the procedure of Folch
+ –
et al. (1957).
Conjugated dienes (CD) were determined according to
the IUPAC method 2.505 (IUPAC, 1992). Pre-weighed
lipid samples were diluted with iso-octane and the absor-
bance at 232 nm was measured with a spectrophotometer
(Shimadzu, Model UV-160A, Tokyo, Japan). Results were
reported as absorbance E1% 1 cm , which was given by the
formula:
Ak
E1%
1 cm ð232Þ ¼ ;
Cd
where Ak is the absorbance at 232 nm, C the concentration
of the lipid solution in iso-octane (g/100 ml) and dis the
length of the cell (cm).
Determination of lipid hydroperoxides was conducted
by the International Dairy Federation standard method
74A:1991 (IDF, 1991) for measurement of peroxide value
(PV), as described by Shantha and Decker (1994). Results
were expressed as milli-equivalents (meq) of hydroperox-
ides/kg of lipids.
Malondialdehyde (MDA) was determined by a selective
third-order derivative spectrophotometric method (Botsog-
lou et al., 1994). Duplicate 1 g samples were mixed with
10 ml of 5% aqueous trichloroacetic acid and 5 ml hexane
containing 0.8% butylated hydroxytoluene (BHT). The
mixture was homogenised with an Ultraturrax (IKA) for
30 s at high speed and was centrifuged for 3 min at 3000g
and the top hexane layer was discarded. A 2.5 ml aliquot
of the bottom aqueous layer was transferred to another
tube and was mixed with 1.5 ml of 0.8% aqueous 2-thiobar-
bituric acid. The mixture was incubated at 70 C for
30 min. Following incubation, the mixture was cooled
under tap water and submitted to conventional spectro-
photometry (Shimadzu, Model UV-160A, Tokyo, Japan)
in the range 400–650 nm. Third-order derivative spectra
were produced by digital differentiation (d3A/dk3, where
A is the absorbance and k is the wavelength) of the normal
spectra obtained at a scanning speed of 480 nm/min, using
a derivative wavelength difference (Dk) setting of 21 nm.
MDA concentration (lg/kg wet tissue) in the samples
was calculated on the basis of the height of the third-order
derivative peak at 521.5 nm by referring to slope and inter-
cept data of the computed least-squares fit of a freshly pre-
pared standard calibration curve.
2.6. Colour evaluation
Burgers were allowed to thaw for 1 h prior to colour
evaluation. Colour of the burgers from each treatment
 D. Georgantelis et al. / Meat Science 75 (2007) 256–264 259

was assessed both visually and by instrumental colour mea- Table 3

surements. Visual assessment was imprinted in high-resolu- Mean fatty acid composition (% of total fatty acids) of the initial meat
batter used for the preparation of the experimental burgers (n = 6)
tion pictures taken on each sampling day. Instrumental
colour was determined using a HUNTERLAB Colorimeter Fatty acid % of total (Mean ± SE)
(L*a*b*) D25 DP9000 (Hunter Associates Laboratory Inc., C 10:0 0.08 ± 0.003
Reston, USA), calibrated to black and white standards. C 12:0 0.11 ± 0.010
C 14:0 3.64 ± 0.021
The tristimulus L*a*b* measurement mode was used as this C 14:1 0.43 ± 0.015
relates to human eye response to colour (Hunter & Harold, C 15:0 0.61 ± 0.008
1987). L*, a* and b* values were measured on five different C 16:0 26.41 ± 0.212
spots on each of two samples. Results were recorded as the C 16:1 trans 0.46 ± 0.018
mean of these measurements. From the measured values, C 16:1 cis 4.51 ± 0.033
C 17:0 1.33 ± 0.028
chroma [(a*2 + b*2)1/2] and hue (b*/a*) were also calculated. C 17:1 0.77 ± 0.008
C 18:0 13.48 ± 0.021
2.7. Statistical analysis C 18:1 trans 2.83 ± 0.092
C 18:1 cis 35.98 ± 0.200
All data were analysed using the General Linear Model C 18:1 x-7 2.22 ± 0.058
C 18:2 x-6 trans 0.47 ± 0.013
of ANOVA with treatment and time as factors, after nor- C 18:2 x-6 cis 2.32 ± 0.038
mality and homogeneity of variances was confirmed. The C 18:3 x-6 0.09 ± 0.003
statistical significance of the differences was checked using C 18:3 x-3 0.94 ± 0.033
the Student–Newman–Keuls (SNK) test at the 0.05 level, C 20:0 0.12 ± 0.008
while correlations between different parameters were evalu- C 20:1 0.11 ± 0.003
C 20:4 x-6 0.54 ± 0.013
ated with the Pearson’s correlation coefficient. All statisti- C 20:5 x-3 0.21 ± 0.010
cal analyses were conducted by use of the SPSS statistical P
package (SPSS 10.05, SPSS Inc., Woking, Surrey, UK). P Saturated 45.78 ± 0.254
MUFA’s 47.31 ± 0.278
P
PUFA’s 4.57 ± 0.078
3. Results and discussion

3.1. Proximate analysis and fatty acid profile of the raw
material
Mean percent contents of protein, fat, moisture and ash
for the meat mixture used for the preparation of experi-
mental burgers are shown in Table 2, while mean fatty acid
composition is presented in Table 3. The targeted value of
ca. 15% for fat content was achieved, while mean fatty acid
composition was found within the ranges reported by
Enser, Hallett, Hewitt, Fursey, and Wood (1996) for beef
intramuscular fat and adipose tissue.
3.2. Lipid oxidation
Concentrations of primary oxidation products in the
lipid fraction of the burgers (CD and PV) together with
those of MDA during the 180-day storage period are pre-
sented in Table 4 and Fig. 1. Values for these parameters
differed significantly (P 6 0.05) between all treatments dur-
ing the whole storage period. Treatments containing chito-
Table 2
Mean percent proximate analysis of the meat mixture used for the
preparation of the experimental burgers (n = 6)
Component (%) Mean ± SE
Protein 18.1 ± 0.15
Moisture 64.4 ± 0.31
Fat 15.2 ± 0.45
Ash 0.9 ± 0.01
san (R + C, T + C, CHI) exhibited the lowest values for all
oxidation products measured. The best antioxidative effect
(P 6 0.05) was obtained by the combination of rosemary
extract and chitosan (R + C), for which the values of CD
and PV were lower at the end of storage period (day 180)
than those obtained as early as day 30 in the controls. Fur-
thermore, samples containing combinations of antioxi-
dants (R + C and T + C) had significantly (P 6 0.05)
lower PV, CD and MDA concentrations than those con-
taining the individual antioxidants (CHI, ROS and
TOC), a fact that indicates the occurrence of a synergistic
effect as regards to prevention of lipid oxidation. Signifi-
cant (P 6 0.01) linear correlations ranging from 0.90 to
0.97 were observed between the PV, CD and MDA con-
tents of the burgers (Table 5).
In agreement with our findings, the positive effects of the
individual use of chitosan, rosemary and a-tocopherol as
regards prevention of lipid oxidation are well documented,
whereas, to the best of our knowledge, there are no reports
on the combined use of chitosan with either rosemary or a-
tocopherol regarding effects on lipid oxidation. Kamil et al.
(2002) observed a 61% decrease in the peroxide value of her-
ring (Clupea harengus) samples containing 200 mg/kg chito-
san, as compared to the controls. In addition, Darmadji and
Izumimoto (1994) reported that the TBA value (expressed
as mg MDA/kg) of beef containing 1% chitosan was at
the same level after 10 days of storage at 4 C as it was on
day 0, whereas the respective value of the control samples
increased sharply. McCarthy, Kerry, Kerry, Lynch, and
Buckley (2001) demonstrated that rosemary added at a level
260 D. Georgantelis et al. / Meat Science 75 (2007) 256–264

Table 4
Peroxide value (PV) and conjugated dienes’ (CD) content during frozen
storage of the experimental burgers for 180 days (n = 6; mean ± standard
error)
Storage Treatment PV (meq CD ðE1%
1 cmð232Þ Þ
time (days) peroxides/kg fat)
0 All treatments 0.24 ± 0.007 2.74 ± 0.045
30 R+C 0.26 ± 0.009a 2.79 ± 0.052a
ROS 0.45 ± 0.008c 3.15 ± 0.046c
T+C 0.35 ± 0.020b 3.01 ± 0.038b
CHI 0.38 ± 0.010b 3.11 ± 0.016c
TOC 0.52 ± 0.010d 3.29 ± 0.054d
CON 0.76 ± 0.021e 3.52 ± 0.063e
60 R+C 0.30 ± 0.012a 2.98 ± 0.037a
ROS 0.66 ± 0.009d 3.42 ± 0.045d
T+C 0.44 ± 0.026b 3.20 ± 0.046b
CHI 0.53 ± 0.023c 3.32 ± 0.022c
TOC 0.76 ± 0.019e 3.62 ± 0.034e
CON 1.43 ± 0.017f 3.98 ± 0.036f
90 R+C 0.45 ± 0.021a 3.20 ± 0.028a Fig. 1. MDA contents of the experimental burgers during frozen storage
ROS 1.05 ± 0.038d 3.79 ± 0.042d for 180 days (n = 6). For treatment description, see Table 1.
T+C 0.54 ± 0.030b 3.33 ± 0.032b
CHI 0.71 ± 0.042c 3.48 ± 0.051c
TOC 1.35 ± 0.043e 4.04 ± 0.030e studied the influence of both chain-breaking [rosemary ole-
CON 2.42 ± 0.108f 4.72 ± 0.053f
120 R+C 0.57 ± 0.024a 3.33 ± 0.037a
orisin and butylated hydroxyanisole/butylated hydroxytol-
ROS 1.27 ± 0.042d 3.92 ± 0.037d uene (BHA/BHT) combination] and preventive
T+C 0.75 ± 0.020b 3.50 ± 0.039b antioxidants (sodium tripolyphosphate) on oxidative stabil-
CHI 0.90 ± 0.026c 3.65 ± 0.028c ity during frozen storage of precooked roast beef slices con-
TOC 1.57 ± 0.035e 4.25 ± 0.033e taining 0.75% (w/w) salt. Their results indicated that
CON 2.86 ± 0.096f 4.91 ± 0.043f
150 R+C 0.66 ±0.023a 3.41 ± 0.024a
sodium tripolyphosphate, which is a known chelating agent,
ROS 1.46 ± 0.042d 4.01 ± 0.038d showed the best antioxidative effect of all combinations
T+C 0.80 ± 0.024b 3.60 ± 0.028b used, while the limited effect of rosemary in that study
CHI 1.00 ± 0.033c 3.73 ± 0.033c was attributed to inability of inhibiting the pro-oxidant
TOC 1.69 ± 0.022e 4.51 ± 0.041e activity of salt. The superiority of antioxidants possessing
CON 3.17 ± 0.063f 5.03 ± 0.070f
180 R+C 0.74 ± 0.035a 3.47 ± 0.041a
chelating activity could explain the overall better perfor-
ROS 1.59 ± 0.043d 4.07 ± 0.024d mance of chitosan compared to rosemary extract and a-
T+C 0.90 ± 0.037b 3.67 ± 0.025b tocopherol in the present study.
CHI 1.07 ± 0.041c 3.77 ± 0.046c Lipid oxidation in the controls was more intense com-
TOC 1.92 ± 0.028e 4.62 ± 0.030f pared to the treated samples; maximum values for PV
CON 2.69 ± 0.115f 4.44 ± 0.057e
and CD contents were reached on day 150, after which a
For treatment description see Table 1. decline was observed. At that point of storage it is possible
*Means in the same column within the same storage day bearing a different
letter (a–f) differ significantly (P < 0.05).
that the rate of hydroperoxide decomposition is higher
than the rate at which they are formed. Similarly, Awad,
Powrie, and Fennema (1968) reported a reduction of PV
in beef meat after two weeks’ storage at 4 C. The shorter
of 0.10% in patties made from previously frozen pork signif- time of occurrence of this reduction compared to our find-
icantly (P 6 0.05) inhibited lipid oxidation, as assessed by ings is obviously related to the different storage tempera-
TBARS values, during 9-day storages at 4 C, while tures. Regarding the MDA contents of the controls, a
TBARS values for the control samples increased almost maximum value of 4814.7 lg/kg was obtained on the
eightfold. Furthermore, Verma and Sahoo (2000) have 180th day of frozen storage. Verma and Sahoo (2000) indi-
shown that addition of 10 ppm a-tocopherol acetate in cate MDA concentrations between 1000 and 2000 lg/kg as
ground chevon meat resulted in limited but significant inhi- threshold values for rancidity, while Greene and Cumuze
bition of lipid oxidation (TBARS and PV) compared to (1982) considered a TBARS range of 600–2000 lg/kg to
control samples after 9 days of refrigerated storage. St. be the minimum detectable level for oxidised flavour in
Angelo, Crippen, Dupuy, and James, (1990) indicated that ground beef by an inexperienced panel. Accordingly, the
the antioxidative effect of a-tocopherol was weaker than control samples in the present study would be perceived
that of rosemary extract in ground beef patties stored at as rancid as early as in the 90th day of storage, TOC and
20 C, while the best results were obtained by using anti- ROS samples at the 120th and 150th day, respectively,
oxidants capable of acting as chelating agents. In the same while samples containing chitosan did not exceed the
context, Murphy, Kerry, Buckley, and Gray (1998) have threshold values until the end of the storage time.
 D. Georgantelis et al. / Meat Science 75 (2007) 256–264 261

Table 5
Pearson correlations between lipid oxidation parameters and instrumental colour measurements for all burgers’ treatments (n = 256)
MDA PV CD L* a* b* Hue Chroma
MDA 1.000
PV 0.967** 1.000
CD 0.905** 0.946** 1.000
L* 0.574** 0.506** 0.423** 1.000
a* 0.863** 0.854** 0.889** 0.506** 1.000
b* 0.707** 0.736** 0.840** 0.223** 0.861** 1.000
Hue 0.887** 0.820** 0.787** 0.662** 0.918** 0.645** 1.000
Chroma 0.826** 0.835** 0.897** 0.416** 0.985** 0.932** 0.844** 1.000
**
P < 0.01.

3.3. Colour evaluation
Appearance of the beef burgers, as assessed visually dur-
ing the 180-day storage period, is presented in Fig. 2. Sam-
ples from treatments containing chitosan, individually or in
combination (CHI, R + C, T + C) had a more intense red
colour than samples containing only rosemary extract
(ROS) and a-tocopherol (TOC) or the controls, which
was obvious from day-90 until the end of the storage per-
iod. Appearance of the latter samples after day-120 was
judged as unacceptable by a group of assessors.
Instrumental measurements of colour during the 180
days of frozen storage are presented in Table 6 and
Fig. 3. A decreasing trend was observed as regards to a*
values (indicative of red colour), chroma (colour intensity)
and b* values (yellow colour), together with an increasing
trend in hue values, which are attributed to the gradual oxi-
dation of myoglobin and accumulation of metmyoglobin
Fig. 2. Appearance of the experimental beef burgers during storage for:
(a) 30 days, (b) 60 days, (c) 90 days, (d) 120 days, (e) 150 days and (f) 180
days. (1) Rosemary + Chitosan; (2) Rosemary; (3) a-tocopherol + Chito-
san; (4) Chitosan; (5) a-tocopherol and (6) Control.
with time (Isdell, Allen, Doherty, & Butler, 2003; Mancini
& Hunt, 2005; Ruiz de Huidobro, Miguel, Onega, & Bláz-
quez, 2003). This is also supported by the strong negative
correlation coefficients between lipid oxidation parameters
and a* values and chroma and positive correlation with hue
(Table 5). Statistical analysis revealed significant (P 6 0.05)
differences between all experimental treatments and con-
trols for a* values, hue and chroma during the whole stor-
age time, while colour stability and retention of red colour
was significantly (P 6 0.05) improved in samples from
treatments containing chitosan (CHI, R + C, T + C). The
best synergistic effect was again obtained from the combi-
nation of chitosan with rosemary extract, whereas individ-
ual use of the chain-breaking antioxidants (rosemary
extract and a-tocopherol) significantly (P 6 0.05) improved
colour stability compared to the controls. Individual use of
the rosemary extract, however, had a poorer effect on col-
our retention compared to a-tocopherol, which is in con-
trast to their respective influence on oxidative stability
(Table 4). Lower a* values have also been reported in pork
patties containing rosemary extract compared to those con-
taining sage or soya proteins after 6 days of refrigerated
(4 C) storage, despite the overall better effect of rosemary
on lipid oxidation compared to sage and soya proteins
(McCarthy et al., 2001). In contrast, Sebranek et al.
(2005) have demonstrated better red colour retention in
raw pork sausages by rosemary extract compared to
BHA/BHT after 84 days of storage. The variation in rose-
mary efficiency between different studies could be attrib-
uted to differences in the oxidation pattern of
oxymyoglobin under conditions of reduced enzymatic
activity, where storage temperature, packaging method,
muscle type and light intensity (photo-oxidation) are major
influencing factors (McDougall, 1982), as well as to differ-
ences in the meat species studied.
Efficiency of dietary a-tocopherol in meat colour reten-
tion has been demonstrated by numerous researchers
(Arnold, Arp, Scheller, Williams, & Schaefer, 1993; Faust-
man, Chan, Schaefer, & Havens, 1998; Mitsumoto,
Arnold, Schaefer, & Cassens, 1993). However, postmortem
supplementation of a-tocopherol at similar concentrations
to those obtained in muscles after in vivo administration
has been reported to have an inferior effect as regards to
colour retention (Mitsumoto et al., 1993). This could
262 D. Georgantelis et al. / Meat Science 75 (2007) 256–264

Table 6
Instrumental colour parameters (L*, b*, hue and chroma) during frozen storage of the experimental burgers for 180 days (n = 6; mean ± standard error)
Storage time (days) Treatment L* b* Hue Chroma
0 R+C 48.59 ± 0.116e 20.75 ± 0.123e 0.93 ± 0.006a 30.39 ± 0.094e
ROS 46.61 ± 0.131b 18.91 ± 0.080a 1.03 ± 0.004c 26.41 ± 0.086a
T+C 47.02 ± 0.066c 19.66 ± 0.038c 0.94 ± 0.002a 28.64 ± 0.038c
CHI 47.61 ± 0.094d 20.17 ± 0.048d 0.94 ± 0.002a 29.43 ± 0.042d
TOC 46.43 ± 0.054ab 19.02 ± 0.046ab 0.98 ± 0.003b 27.13 ± 0.080b
CON 46.19 ± 0.041a 19.17 ± 0.047b 1.00 ± 0.003b 27.05 ± 0.043b
30 R+C 46.15 ± 0.194d 19.37 ± 0.061e 0.92 ± 0.004b 28.61 ± 0.092e
ROS 46.31 ± 0.166d 17.40 ± 0.097b 1.11 ± 0.012c 23.47 ± 0.107b
T+C 45.66 ± 0.171c 18.56 ± 0.052d 0.88 ± 0.007a 28.08 ± 0.125d
CHI 45.35 ± 0.209b 19.86 ± 0.077f 0.94 ± 0.005b 28.97 ± 0.130f
TOC 45.70 ± 0.061c 17.64 ± 0.060c 1.12 ± 0.012c 23.67 ± 0.076c
CON 45.06 ± 0.114a 17.22 ± 0.076a 1.13 ± 0.008c 23.03 ± 0.115a
60 R+C 47.04 ± 0.060c 18.74 ± 0.043e 0.98 ± 0.002a 26.81 ± 0.038e
ROS 46.81 ± 0.105c 17.09 ± 0.034b 1.40 ± 0.005c 20.98 ± 0.035b
T+C 46.42 ± 0.198b 18.33 ± 0.029d 0.98 ± 0.002a 26.26 ± 0.052d
CHI 46.17 ± 0.045a 18.29 ± 0.072d 0.96 ± 0.003a 26.40 ± 0.063d
TOC 46.15 ± 0.070a 17.41 ± 0.046c 1.19 ± 0.006b 22.01 ± 0.038c
CON 46.26 ± 0.080ab 16.47 ± 0.098a 1.44 ± 0.011d 20.06 ± 0.083a
90 R+C 45.89 ± 0.062a 18.11 ± 0.037d 0.98 ± 0.004a 25.90 ± 0.038e
ROS 46.97 ± 0.165d 16.51 ± 0.074b 1.65 ± 0.009d 19.29 ± 0.066b
T+C 46.61 ± 0.123bc 18.36 ± 0.190e 1.04 ± 0.008b 25.50 ± 0.192d
CHI 46.54 ± 0.043b 17.94 ± 0.125c 1.00 ± 0.013a 25.35 ± 0.090d
TOC 46.38 ± 0.276b 16.59 ± 0.067b 1.50 ± 0.012c 19.93 ± 0.056c
CON 46.84 ± 0.085cd 16.09 ± 0.080a 1.70 ± 0.011e 18.66 ± 0.078a
120 R+C 46.75 ± 0.114ab 17.66 ± 0.039c 0.98 ± 0.005a 25.20 ± 0.051e
ROS 47.75 ± 0.210d 16.31 ± 0.041ab 1.89 ± 0.019f 18.45 ± 0.064a
T+C 46.53 ± 0.115a 17.59 ± 0.246c 1.09 ± 0.016c 23.94 ± 0.190c
CHI 46.84 ± 0.086b 17.88 ± 0.109d 1.04 ± 0.013b 24.81 ± 0.054d
TOC 47.68 ± 0.230d 16.33 ± 0.028b 1.83 ± 0.011d 18.62 ± 0.024b
CON 47.17 ± 0.080c 16.15 ± 0.075a 1.86 ± 0.017e 18.33 ± 0.054a
150 R+C 46.24 ± 0.074a 17.22 ± 0.038d 1.07 ± 0.005a 23.55 ± 0.040d
ROS 48.17 ± 0.125d 16.43 ± 0.023b 2.66 ± 0.011e 17.55 ± 0.026b
T+C 46.35 ± 0.105a 16.99 ± 0.091c 1.17 ± 0.006c 22.36 ± 0.083c
CHI 46.71 ± 0.078b 17.87 ± 0.095e 1.11 ± 0.006b 24.09 ± 0.082e
TOC 47.67 ± 0.034c 16.14 ± 0.040a 2.37 ± 0.017d 17.52 ± 0.047b
CON 48.42 ± 0.062e 16.27 ± 0.078ab 2.68 ± 0.014e 17.36 ± 0.077a
180 R+C 46.52 ± 0.074b 16.65 ± 0.024cd 1.11 ± 0.011a 22.40 ± 0.078e
ROS 48.66 ± 0.068d 16.73 ± 0.043d 2.81 ± 0.011d 17.76 ± 0.043c
T+C 46.06 ± 0.065a 16.22 ± 0.054b 1.25 ± 0.005b 20.78 ± 0.062d
CHI 46.62 ± 0.090b 16.54 ± 0.057c 1.11 ± 0.008a 22.29 ± 0.104e
TOC 48.17 ± 0.141c 15.69 ± 0.041a 2.66 ± 0.011c 16.77 ± 0.039a
CON 48.83 ± 0.060d 16.37 ± 0.080b 2.99 ± 0.015e 17.26 ± 0.082b
For treatment description see Table 1.
*
Means in the same column within the same storage day bearing a different letter (a–f) differ significantly (P 6 0.05).

explain the relatively poor colour retention of a-tocopherol
compared to the controls in the present study, despite the
observed difference of approximately 30% in MDA con-
tents at the end of the storage period.
In agreement with our findings, chitosan has been also
shown to have an important effect in red colour retention
by other researchers. Darmadji and Izumimoto (1994) have
reported higher a* values in minced beef meat containing
0.2%, 0.5% and 1% chitosan after 10 days of refrigerated
storage. Youn, Park, Kim, and Ahn (1999) have also stud-
ied the effect of chitosan on the colour of pork sausages
stored at 4 C and found that a 50% reduction of nitrites
had no influence on the colour of sausages containing
0.2% chitosan.
As regards to red colour retention between samples
containing combinations of chain-breaking and preventive
antioxidants, Youn et al. (1999) have shown that the best
effect was obtained in pork sausages by the combination
of rosemary and chitosan. Djenane, Sánchez-Escalante,
Beltrán, and Roncalés (2002) studied the effects of a-
tocopherol, taurine and rosemary in combination with
vitamin C on the colour of beef steaks and have con-
cluded that both combinations of vitamin C with rose-
mary and taurine resulted in significantly (P 6 0.05)
higher a* values than the combination with a-tocopherol
or the controls.
Initial measurements (day 0) of b* values were higher
in samples from treatments containing chitosan, which
could be attributed to the natural yellowish colour of
chitosan affecting the burgers’ colour. Similar observa-
tions have been also made by Youn et al. (1999) and Jo
et al. (2001) in pork sausages with added chitosan. As
 D. Georgantelis et al. / Meat Science 75 (2007) 256–264
Fig. 3. Instrumental colour a* values of the experimental burgers during
frozen storage for 180 days (n = 6). For treatment description, see Table 1.
regards to changes in L* values during storage, there was
an increasing trend in samples not containing chitosan
(ROS, TOC and CON), which could be the result of grad-
ual protein decomposition, leading to increase of light
scattering (McDougall, 1982). On the other hand, L* val-
ues during storage of samples containing chitosan (CHI,
R + C and T + C) showed a slightly decreasing trend. A
possible explanation could be the moisture retaining abil-
ity of chitosan (Cho, No, & Meyers, 1998), which could
repress drip loss of meat during thawing, resulting in
increased transparency and lowering of lightness. A simi-
lar trend in minced meat containing chitosan compared to
controls was noted by Darmadji and Izumimoto (1994),
whereas Jo, Lee, Lee, and Byun (2001) reported an
increasing trend in L* values of samples containing
chitosan.
4. Conclusions
Results of the present study demonstrate the positive
effects of chitosan, added individually or in combination
with rosemary or a-tocopherol, on both retarding lipid oxi-
dation and improving colour retention of beef burgers dur-
ing frozen storage (18 C) for 180 days. The best results
were obtained with the combination of rosemary with
chitosan. This combination could have for commercial
use to improve preservation of beef burgers. Further
research could investigate the combined application of
rosemary and chitosan in different meat products as well
as the use of different quantities to those used in this study
for optimisation of the antioxidative effects.
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