Racanicci, 2007

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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: [email protected]

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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


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Racanicci, 2007