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O R I G I N A L P A P E R
Mate ( Ilex paraguariensis) as a source of water extractableantioxidant for use in chicken meat
Aline M. C. Racanicci Bente Danielsen Leif H. Skibsted
Received: 12 April 2007 / Revised: 19 June 2007/ Accepted: 24 June 2007
Springer-Verlag 2007
Abstract Aqueous extract of mate, made from dried
leaves of Ilex paraguariensis, St. Hilaire, was shown to beeffective during chilled storage for up to 10 days in pro-
tecting lipids and vitamin E against oxidation in pre-
cooked meat balls made from chicken breast added 0.5%
salt and packed in atmospheric air. Extracts made with
water, methanol, ethanol or 70% aqueous acetone were
evaluated by comparing (1) total phenolic content, (2)
radical scavenging capacity, (3) effect on lipid oxidation in
a food emulsion model, and in liposomes. Based on the
three-step evaluation, aqueous mate extract was preferred
for food use. Dried leaves were further compared to dried
rosemary leaves in chicken meat balls, and mate (0.05 and
0.10%) found to yield equal or better protection thanrosemary at the same concentration against formation of
secondary lipid oxidation products.
Keywords Mate Antioxidant capacity ESR TBARS Chicken meat stability
Introduction
Herbs and spices other than the widely used rosemary are
currently being explored for protection of processed food
sensitive to lipid oxidation [1, 2]. Pre-cooked chicken meat
is an example of easily oxidized food for which significantquality improvements have been obtained by herb addition
at a sensory acceptable level [3]. Chicken meat is becoming
increasingly important world-wide and in Brazil, the pro-
duction increases rapidly and different waste materials from
local herb and vegetable productions are considered as a new
antioxidant source for protection of chicken meat products.
Mate, dried leaves of Ilex paraguariensis, St. Hilaire, native
of and cultivated in Brazil, Argentina, Uruguay, and Para-
guay, is used to prepare an infusion important to the region
as a bitter taste stimulant. Mate is generally accepted for
human consumption and is known to have a high content of
phenols [4], and was accordingly evaluated for protection of pre-cooked chicken meat using a four-step evaluation pro-
tocol [5]. In the present investigation, extraction efficiency
of different solvents for potential antioxidants was com-
pared prior to determination of antioxidant effect in perox-
idating lipids in model systems. The final evaluation of mate
as an antioxidant for food use included storage experiments
with pre-cooked meat balls added mate extract or added
dried leaves in comparison with dried rosemary.
Material and methods
Mate samples and extracts
Mate (3.0 kg of pure dried leaves of Ilex paraguariensis,
St. Hilaire) of a brazilian trade mark (Vier Industria e
Comercio do Mate Ltda, Santa Rosa, RS, Brazil) was
purchased at the local market in Porto Alegre, Rio Grande
do Sul, Brazil. Three extractions were carried out at the
same day as analysis by mixing 0.5 g of mate in 50 mL of
solvent (water, methanol, ethanol or 70% aqueous acetone)
A. M. C. Racanicci
Department of Animal Science, Escola Superior de Agricultura
‘‘Luiz de Queiroz’’—ESALQ, University of Sao Paulo,
Avenida Padua Dias, 11, CEP 13418-900 Piracicaba, SP, Brazil
B. Danielsen L. H. Skibsted (&)
Department of Food Science, Food Chemistry,
University of Copenhagen, Rolighedsvej 30,
1958 Frederiksberg C, Denmark
e-mail: ls@life.ku.dk
1 3
Eur Food Res Technol
DOI 10.1007/s00217-007-0718-5
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followed by sonicating for 10 min in an ultrasonic bath.
The suspensions were centrifuged for 15 min at 3,000 rpm
and filtrated. The extracts were kept at 4 C until use. For
the storage experiment, mate was ground in a mortar and
added to the meat as dried leaves (0.05 or 0.10%) or as an
aqueous extract corresponding to the same content of dried
leaves. The water used was purified on a Milli-Q purifi-
cation train (Millipore Corp., Bedford, MA, USA). Theother solvents were of analytical grade.
Total phenolic content
The amount of total phenolics was determined by the
procedure of Folin–Ciocalteau described by Amerine and
Ough [6]. Extracts of mate in water, methanol, ethanol or
70% aqueous acetone (0.5 mL, three replicates) were
mixed with 30 mL of Milli-Q water and 2.5 mL of Folin–
Ciocalteu’s reagent (Merck 9001, Darmstadt, Germany).
After 30 s, 7.5 mL of 20% sodium carbonate solution wasadded and the solution was mixed and diluted with water to
a final volume of 50 mL. After 2 h in the dark at 20 C, the
absorbance of the samples was measured at 765 nm using a
Shimadzu UV-1200 spectrophotometer (Shimadzu, Kyoto,
Japan). The phenolic content was expressed in mg of gallic
acid equivalent (GAE) per liter of extract and in mg per
gram of sample. The standard curve (50–750 mg L1) was
based on analytical grade gallic acid (Sigma-Aldrich,
Steinheim, Germany).
Oxygen consumption assay in emulsions
The rate of depletion of oxygen was measured based on the
metmyoglobin (MMb) initiated oxidation of methyl lino-
leate as described by Hu and Skibsted [7]. The 250 lL of
methyl linoleate (28.2 mM, dissolved in methanol) was
mixed with 62.5 lL Tween-20 (0.04 g mL1, dissolved in
methanol) and the methanol was removed under a nitrogen
flow. This procedure was followed by the addition of
2.50 mL of 5.00 mM thermostatted (25 C) air-saturated
phosphate buffer (pH 6.8) and 10 lL of mate extracts
(water, methanol, ethanol or 70% aqueous acetone). The
water extract was also tested at addition levels of 2.5, 5.00,
and 7.50 lL in addition to 10 lL. In order to initiate oxi-
dation, 25 lL of MMb aqueous solution (0.20 mM of Type
II horse heart MMb from Sigma, St. Louis, MO, USA) was
added and the sample was immediately transferred to a
70 lL thermostatted (25.0 ± 0.1 C) measuring cell with
no headspace (Chemiware, Viby J., Denmark) to start
oxygen concentration measurements. The relative oxygen
concentration was measured using a Clark electrode con-
nected to a multi-channel analyses ReadOx-4H (Sable
Systems, Henderson, NV, USA) and recorded every 10 s
during 20 min. The electrode was calibrated by a two-point
calibration procedure with anoxic solution and air-saturated
buffer thermostatted at 25 C. The initial oxygen con-
sumption rate V(O2) in lmol1 s1 was calculated using
[O2]initial = 2.6 · 104 mol L1 (water saturated with air at
25 C):
V O2ð Þ ¼ slope O2½ initial 106=100:
The slope (percent of O2 per second) was calculated
from the oxygen consumption in the 80–40% interval
relative to the initial 100% oxygen concentration
corresponding to water saturated with air. The influence
of mate extracts on the initial rate of oxygen consumption
was expressed as an antioxidative index ( I oxygen) relative to
the rate obtained in the absence of the extracts:
I oxygen ¼ V O2ð Þ with mate extract
V O2ð Þ
without mate extract:
Antioxidant activity in liposomes
The preparation of liposomes, initiation of peroxidation of
phospholipids in the liposomes and the measurement of
conjugated dienes were performed as described by Roberts
and Gordon [8] with some modifications. Lipid suspension
was produced using 2.0 mL of soybean L -a-phosphatidyl-
choline (PC from Sigma-Aldrich) solution (0.75 mM in
chloroform) in a 25 mL flask covered with aluminum foil
to avoid light-induced oxidation. The solvent was removed
under reduced pressure on a rotary evaporator (Rotavapor
R-144, Buchi, Flawil, Switzerland) with a vacuum pump(Julabo F25, Seelbach, Germany) in a water bath (Water-
bath B-840, Buchi) set at 30 C, and nitrogen was intro-
duced in the system to re-establish atmospheric pressure.
The lipid residue was rehydrated using 10 mL of phosphate
buffer (0.01 M, pH 6.8) with different concentrations of
mate aqueous extracts, flushed with nitrogen, and quickly
sealed with a cap before it was vortex-mixed for 10 min
and then sonicated in an ultrasonic bath for 30 s to produce
a homogeneous suspension of multi-lamellar liposomes.
Large unilamellar liposomes were obtained by transferring
the liposome suspension to a small volume extrusion
device (Avestin Lipsofast Basic, Avestin, Mannheim,
Germany). The suspension was passed 20 times through a
double layer of polycarbonate membranes (100 nm pore
diameter). Unilamellar liposome suspension with mate
water extracts (2.475 mL) was pipetted into quartz cuvettes
and incubated for 10 min at 37 C within the water-jacket
regulated cell holder of a Shimatzu UV-vis scanning
spectrophotometer model 2101 (Kyoto, Japan). Phospho-
lipid peroxidation was initiated by adding 25 lL
of 2,20azobis-(2-aminopropane)dihydrochloride (AAPH)
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solution (0.75 nM) to each cuvette. The cuvettes were
sealed to prevent evaporation and inverted twice. The
absorbance at 234 nm was measured every 10 min during
900 min for each cuvette against the blank of phosphate
buffer. Up to six sample liposome suspension (two mate
concentrations and three replicates) and one blank lipo-
some suspension were measured in each run.
Electron spin resonance (ESR) spectroscopy assay
based on reduction of Fremy’s salt radical
The antioxidant capacity of mate aqueous extracts on
scavenging the stable radical from Fremy’s salt
(K 2(SO3)2NO) was evaluated according to Rødtjer et al. [9].
Aqueous extracts of mate were mixed with 3.00 mL of
water and 30 lL of Fremy’s salt (700 lM) dissolved in
25% saturated sodium carbonate solution. The concentra-
tion of the Fremy’s salt solution was adjusted based on
spectrophotometric measurements. Three microliters of water and 30 lL of Fremy’s salt corresponded to the blank
solution and represented the total concentration of Fremy’s
salt without addition of the antioxidant. The experiments
consisted of the addition of different concentrations of mate
aqueous extracts to evaluate the reduction of the ESR signal
of Fremy’s salt. The ESR spectra were recorded with a Jeol
JES-FR 30 ESR spectrometer (JEOL Ltd., Tokyo, Japan)
5 min after mixing. The measurements were carried out at
room temperature with the following settings: microwave
power: 4 mW, center field: 336.246 mT, sweep width:
5 mT, sweep time: 2 min, modulation width: 0.10 mT,
amplitude: 790, conversion time: 0.3 s. The intensity of the
ESR signal was measured as the height of the central line of
the peak relative to the height of a Mn(II)-marker attached
to the cavity of the spectrometer. The antioxidant capacity
was calculated on the basis of a linear regression of results
from experiments with up to ten different concentrations of
the samples of mate aqueous extracts. The antioxidant
capacity was expressed as nmol Fremy’s radicals reduced
by nmol of GAE extracted from mate.
Preparation and storage of meat balls
Fresh chicken breast meat produced by Rose Poultry A/S
Denmark (Vinderup, Denmark) was chopped, minced,
weighed, mixed with 0.50% of food grade salt (Danish Salt
I/S, Mariager, Denmark) was added aqueous mate extract
corresponding to 0.05 or 0.10% of dried mate (storage
experiment 1) or with 0.050 or 0.10% of mate or of rosemary
dried leaves made from fresh leaves (Christen Olsen,
Thorslunde, Denmark) by drying at 40 C for 65 h (storage
experiment 2). The control for the meat balls with mate
aqueous extract (experiment 1) were added equivalent vol-
ume of water. Meat balls weighing 30 ± 0.5 g were vac-
uum-packed in bags with low-oxygen permeability and
cooked in boiling water at 100 C for 8 min. The five types
of meat balls studied were accordingly meat balls with each
of the two types of spices, each in two concentrations plus a
control with only salt added. The bags with meat balls were
cooled on ice and then repacked in polyethylene (PE) bagswith high-oxygen transfer rate (2,000 mL/m2
· 24 h ·
atm) and stored in the dark in a cold room at 5 C with
temperature registration for 10 days. Two meat balls from
each of five treatments were analyzed in duplicate on days 0,
1, 3, 6, 8, and 10 of storage for secondary lipid oxidation
products (as TBARS). Vitamin E was analyzed in the stor-
age experiment 1 with aqueous extract of mate on the same
days. Prior to storage (day 0), two samples of fresh and pre-
cooked meat were analyzed in duplicates for total fat
by extraction yielding 1.52 ± 0.01 and 1.65% ± 0.03,
respectively.
Thiobarbituric acid reactive substances (TBARS)
TBARS were assessed according to Madsen et al. [10].
Fifteen microliters of TCA solution (7.5% of trichloro-
acetic acid, 0.1% of EDTA, and 0.1% of propylgallate, all
from Merck) were added to 5.00 g of stored chicken meat
and mixed during 45 s and 13,500 rpm in an Ultra-Turrax
T-25 (Janke & Kunkel IKA-Labortechnik, Staufen,
Germany) and filtrated. Five microliters of the filtrate was
mixed with 5.00 ml of 0.020 M of the TBA (2-thiobarbi-
turic acid from Merck) solution, and the reaction mixture
placed in a water bath at 100 C for 40 min. Absorbance
was measured at 532 and 600 nm using a Shimadzu UV-
1200 Spectrophotometer (Shimadzu) and the difference
(A532–A600 nm) was used in order to correct the absorbance
for turbidity. TBARS were measured in duplicate (exper-
iment 1) and triplicate (experiment 2) and expressed in
lmoles of malondialdehyde (MDA) per kilogram of meat
using a standard curve (0.1–6.0 nM) made with 1,1,3,3-
tetraethoxypropane (TEP from Merck).
Vitamin E
The amount of a-tocopherol in chicken breast meat was
determined using analytical grade chemicals as described
by Jensen et al. [11]. Two samples of each treatment were
homogenized with 20 ml of 1.15% KCl (Merck) solution
using the Ultra Turrax T-25 for 30 s at 13,500 rpm. Two
microliters of the homogenate were transferred to a tube
containing 0.200 ml of saturated KOH (Merck) and
2.00 ml of 0.5% pyrrogalol (Aldrich, Milwaukee, MI,
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USA) in ethanol and weighed and mixed using a vortex
mixer. The samples were saponified in a water bath at
70 C and in darkness for 30 min and lipids were extracted
using hexane + BHT (2,6-di-tert -butyl-p-hydroxytoluene
from Merck) and then centrifuged at 2,500 rpm for 5 min.
(Mistral 2000, Radiometer, Bagsvaerd, Denmark). The
hexane phase was evaporated under nitrogen flow to dry-
ness, and samples were redisolved in 0.25 ml of etha-nol + BHT and quantified by reverse phase HPLC. The
HPLC-system 1100 (Agilent Technologies Inc., Palo Alto,
CA, USA) was connected to a fluorescence detector (HP
1100—G1321A FLD, Agilent Technologies Inc.). Excita-
tion was at 288 nm, emission at 330 nm, and integration
was performed by the HP chemstation using the LC soft-
ware. The column was a 125 · 4 mm2, 5 lm Hypersil
ODS, (Agilent Technologies Inc.). The mobile phase con-
sisted of methanol: water (94:6) at a flow rate of 1.0 mL
min1. Vitamin E was measured in duplicate in experiment
1 and expressed in lg tocopherol per gram of meat sample
using an external standard curve made for a-tocopherol.
Statistical Analysis
The experimental factors in both storage experiments:
treatment (addition of mate aqueous extract in experiment
1 or dried spices in experiment 2) and storage time (days 0,
1, 3, 6, 8, and 10) and the interaction between factors were
studied in a completely randomized design by General
Linear Model Procedure (SAS Version 8.00, Institute Inc.,
Cary, NC, USA). TBARS and vitamin E were the response
variables analyzed in two replications each.
Results and discussion
Mate was found to have a high content of phenolics. These
compounds are known to be caffeoyl derivatives (caffeic
acid, chlorogenic acid, 3,4-dicaffeoylquinic acid, 3,5-dic-
affeoylquinic acid, and 4,5-dicaffeoylquinic acid) and
flavonoids (quercetin, rutin, and kaempferol) and the
presence of phenylpropanoid compounds is strongly related
to the antioxidant properties of plant extracts [12]. Among
the solvents tested, 70% aqueous acetone was found to be
the most efficient for extraction of phenolics as seen from
the analytical results in Table 1. Water was, however,
comparable, and superior to methanol and ethanol, and
water should be preferred as solvent for food use. The
radical scavenging capacity of the aqueous extract as used
for the food studies was further determined by reaction
with Fremy’s salt. As shown in Fig. 1, the ESR signal is
diminished upon addition of Fremy’s salt and the ratio
between the Fremy’s salt reduced and the GAE present in
the aqueous extracts is 3.3 ± 0.l, as shown in Fig. 2. A ratio
close to three shows that reaction between Fremy’s salt and
the extract is to be considered as a titration, since each
gallic acid has three phenolic groups, which may donate a
hydrogen atom to Fremy’s salt.
The next step in the evaluation was to investigate the
effect of mate extract on a peroxidating lipid system. As
shown in Table 2, the oxygen consumption decreased with
increasing addition of aqueous extract of mate. The an-
tioxidative index I oxygen shows an almost linear response tothe mate extract added to peroxidating lipid emulsion. The
ethanol and methanol extracts are less efficient, while 70%
aqueous acetone has the highest effect. The efficiency of
aqueous extract to suppress lipid oxidation was further
confirmed in liposomes, where aqueous extract resulted in
a significant lag-phase, when monitoring oxidation by
formation of conjugated dienes (Fig. 3). Also for the
liposome system, the effect was found to depend on the
amount of extract added.
The food protection study was designed on the basis of
(1) the determination of phenolic compounds in mate, (2)
the demonstration of good radical scavenging capacity, and(3) capability to suppress lipid oxidation in emulsions and
liposomes. Aqueous extract and dried leaves were selected
for the practical test. In experiment 1, the addition of
aqueous extract of mate to pre-cooked meat balls prior to
heat treatment showed a clear effect even at the lowest
concentration (corresponding to 0.05% of dried leaves)
yielding a significant (P < 0.0001) protection against the
formation of secondary lipid oxidation products (Fig. 4).
In order to compare with a well-established protection
strategy, the dried mate leaves were compared to dried
rosemary leaves at the same two levels of addition in
experiment 2. As shown in Fig. 5, the dried mate leaves
were effective and demonstrate stronger antioxidant pro-
tection (P < 0.0001) than rosemary in this specific product.
Interaction between polyphenols and vitamin E in foods
is getting increasing attention [1, 2, 13], and vitamin E was
analyzed during storage in experiment 1, where aqueous
extract was used. Mate added as an aqueous extract pro-
tected not only the lipids against oxidation but also vitamin
E was less oxidized (P < 0.0004) in the product with the
extract added (Fig. 6).
Table 1 Total phenol content of extracts of mate using different
solvents (n = 3)
Mg GAE g1
dried leavesa
Water 83 ± 1
Methanol 42 ± 4
Ethanol 25 ± 270% aqueous acetone 97 ± 8
a GAE gallic acid equivalent from Folin–Ciocalteu method
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interphase between lipids and the aqueous phase of the
meat. Development of new chicken meat products for
marketing will, however, depend on sensory evaluation of
products with mate added and such studies are currently
being conducted.
Acknowledgments This research was sponsored by the OFORSK
Committee, as part of the research program ‘‘FOODANTIOX-New
Antioxidant Strategies for Food Quality and Consumer Health.’’ The
authors thank FAPESP (The State of Sao Paulo Research Foundation)
for financial support such as travel and housing grant.
References
1. Nissen LR, Maansson L, Bertelsen G, Huynh-Ba T, Skibsted LH(2000) J Agric Food Chem 48:5548–5556
2. Bragagnolo N, Danielsen B, Skibsted LH (2007) Innov Food Sci
Technol 8:24–27
3. Racanicci AMC, Danielsen B, Menten JFM, Regitano-dArce
MAB, Skibsted LH (2004) Eur Food Res Technol 218:521–524
4. Filip R, Lopez P, Giberti G, Coussio J, Ferraro G (2001) Fito-
terapia 72:774–778
5. Becker EM, Nissen LR, Skibsted LH (2004) Eur Food Res
Technol 219:561–571
6. Amerine MA, Ough CS (1980) In: Wiley J (ed) Methods for
analysis of must and wines. Wiley-Interscience Publication, New
York, pp 181–184
7. Hu M, Skibsted LH (2002) Food Chem 76:327–333
8. Roberts WG, Gordon MH (2003) J Agric Food Chem 51:1486–
14939. Rødtjer A, Skibsted LH, Andersen ML (2006) Food Chem 99:6–
14
10. Madsen HL, Sørensen B, Skibsted LH, Bertelsen G (1998) Food
Chem 63:173–180
11. Jensen C, Guidera J, Skovgaard IM, Staun H, Skibsted LH,
Jensen SK, Møller AJ, Buckley J, Bertelsen G (1997) Meat Sci
55:491–500
12. Filip R, Ferrato GE (2003) Eur J Nutr 42:50–54
13. Bragagnolo N, Danielsen B, Skibsted LH (2005) Eur Food Sci
Technol 221:610–615
0
50
100
150
200
250
300
control 125 250 500 1.000 3.000
Antioxidant concentration (mg dried leaves mL-1
)
I P ( m i n )
Fig. 3 Induction Periods ( IP) at 30 C for formation of conjugated
dienes in phosphatidyl choline liposomes suspension with different
concentration of mate added as water extracts
0
10
20
30
40
50
60
70
80
0 1 8 10
Days of storage
T B A R S ( u m o l M D A / k g m e a t )
63
Fig. 4 Formation of secondary lipid oxidation products in pre-
cooked chicken meat balls with and without addition of mate aqueous
extract—experiment 1 ( filled square, control; filled circle, mate
extract corresponding to 0.05% of dried leaves; open circle, mate
extract corresponding to 0.10% of dried leaves) during storage at 5 C
measured as thiobarbituric acid reactive substances (lmol malonaldi-
aldehyde kg1 of meat). Mean values of two meat balls with two
repetitions each
0
0
10
20
30
40
50
60
70
80
90
10
Days of s torage
T B A R S ( u m o l M D A / k g m e a t )
1 3 6 8
Fig. 5 Formation of secondary lipid oxidation products in pre-
cooked chicken meat balls with and without addition of spices—
experiment 2 ( filled square, control; filled triangle, rosemary 0.05%
of dried leaves; open triangle, rosemary 0.10% of dried leaves; filled
circle, mate 0.05% of dried leaves; open circle, mate 0.10% of dried
leaves) during storage at 5 C measured as thiobarbituric acid reactive
substances (lmol malonaldialdehyde kg1 of meat). Mean values of
two meat balls with three repetitions each
0,00
1,00
2,00
3,00
4,00
5,00
10
Days of storage
V i t . E
( u g a l f a - t o c o f e r o l / g m e a t )
0 1 3 6 8
Fig. 6 Effect of mate aqueous extracts ( filled square, control; filled
circle, mate extract corresponding to 0.05% of dried leaves; open
circle, mate extract corresponding to 0.10% of dried leaves) prior to
cooking on the concentration of vitamin E (lg a-tocopherol g1 of
meat) in chicken meat balls during storage at 5 C. Mean values of
two meat balls and two repetitions each
Eur Food Res Technol
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