Eur Food Res Technol DOI 10.

1007/s00217-007-0718-5

ORIGINAL PAPER

Mate (Ilex paraguariensis) as a source of water extractable antioxidant 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 be effective during chilled storage for up to 10 days in protecting lipids and vitamin E against oxidation in precooked 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 than rosemary 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 signicant quality 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 production 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 Paraguay, 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 protocol [5]. In the present investigation, extraction efciency of different solvents for potential antioxidants was compared prior to determination of antioxidant effect in peroxidating lipids in model systems. The nal 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

A. M. C. Racanicci Department of Animal Science, Escola Superior de Agricultura o Paulo, Luiz de QueirozESALQ, University of Sa dua Dias, 11, CEP 13418-900 Piracicaba, SP, Brazil Avenida Pa 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

Mate (3.0 kg of pure dried leaves of Ilex paraguariensis, stria e St. Hilaire) of a brazilian trade mark (Vier Indu rcio do Mate Ltda, Santa Rosa, RS, Brazil) was Come 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)

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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 ltrated. 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 puried on a Milli-Q purication train (Millipore Corp., Bedford, MA, USA). The other solvents were of analytical grade.

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 consumption rate V(O2) in lmol1 s1 was calculated using [O2]initial = 2.6 104 mol L1 (water saturated with air at 25 C): VO2 slope O2 initial 106 =100: The slope (percent of O2 per second) was calculated from the oxygen consumption in the 8040% interval relative to the initial 100% oxygen concentration corresponding to water saturated with air. The inuence of mate extracts on the initial rate of oxygen consumption was expressed as an antioxidative index (Ioxygen) relative to the rate obtained in the absence of the extracts: Ioxygen VO2 with mate extract : VO2 without mate extract

Total phenolic content The amount of total phenolics was determined by the procedure of FolinCiocalteau 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 Ciocalteus reagent (Merck 9001, Darmstadt, Germany). After 30 s, 7.5 mL of 20% sodium carbonate solution was added and the solution was mixed and diluted with water to a nal 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 (50750 mg L1) was based on analytical grade gallic acid (Sigma-Aldrich, Steinheim, Germany).

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 modications. Lipid suspension was produced using 2.0 mL of soybean L-a-phosphatidylcholine (PC from Sigma-Aldrich) solution (0.75 mM in chloroform) in a 25 mL ask covered with aluminum foil to avoid light-induced oxidation. The solvent was removed under reduced pressure on a rotary evaporator (Rotavapor chi, Flawil, Switzerland) with a vacuum pump R-144, Bu (Julabo F25, Seelbach, Germany) in a water bath (Water chi) set at 30 C, and nitrogen was introbath B-840, Bu 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, ushed 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). Phospholipid peroxidation was initiated by adding 25 lL of 2,20 azobis-(2-aminopropane)dihydrochloride (AAPH)

Oxygen consumption assay in emulsions The rate of depletion of oxygen was measured based on the metmyoglobin (MMb) initiated oxidation of methyl linoleate 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 ow. 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 oxidation, 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 connected to a multi-channel analyses ReadOx-4H (Sable

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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 liposome suspension were measured in each run.

Electron spin resonance (ESR) spectroscopy assay based on reduction of Fremys salt radical The antioxidant capacity of mate aqueous extracts on scavenging the stable radical from Fremys salt (K2(SO3)2NO) was evaluated according to Rdtjer et al. [9]. Aqueous extracts of mate were mixed with 3.00 mL of water and 30 lL of Fremys salt (700 lM) dissolved in 25% saturated sodium carbonate solution. The concentration of the Fremys salt solution was adjusted based on spectrophotometric measurements. Three microliters of water and 30 lL of Fremys salt corresponded to the blank solution and represented the total concentration of Fremys 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 Fremys 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 eld: 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 Fremys radicals reduced by nmol of GAE extracted from mate.

aqueous extract (experiment 1) were added equivalent volume of water. Meat balls weighing 30 0.5 g were vacuum-packed in bags with low-oxygen permeability and cooked in boiling water at 100 C for 8 min. The ve 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) bags with 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 ve 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 storage experiment 1 with aqueous extract of mate on the same days. Prior to storage (day 0), two samples of fresh and precooked 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 trichloroacetic 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 ltrated. Five microliters of the ltrate was mixed with 5.00 ml of 0.020 M of the TBA (2-thiobarbituric 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 UV1200 Spectrophotometer (Shimadzu) and the difference (A532A600 nm) was used in order to correct the absorbance for turbidity. TBARS were measured in duplicate (experiment 1) and triplicate (experiment 2) and expressed in lmoles of malondialdehyde (MDA) per kilogram of meat using a standard curve (0.16.0 nM) made with 1,1,3,3tetraethoxypropane (TEP from Merck).

Preparation and storage of meat balls Vitamin E 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 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 saponied 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 ow to dryness, and samples were redisolved in 0.25 ml of ethanol + BHT and quantied by reverse phase HPLC. The HPLC-system 1100 (Agilent Technologies Inc., Palo Alto, CA, USA) was connected to a uorescence detector (HP 1100G1321A FLD, Agilent Technologies Inc.). Excitation was at 288 nm, emission at 330 nm, and integration was performed by the HP chemstation using the LC software. The column was a 125 4 mm2, 5 lm Hypersil ODS, (Agilent Technologies Inc.). The mobile phase consisted of methanol: water (94:6) at a ow 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-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid) and avonoids (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 efcient 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 Fremys salt. As shown in Fig. 1, the ESR signal is diminished upon addition of Fremys salt and the ratio between the Fremys salt reduced and the GAE present in the aqueous extracts is 3.3 0.l, as shown in Fig. 2. A ratio

Table 1 Total phenol content of extracts of mate using different solvents (n = 3) Mg GAE g1 dried leavesa Water Methanol Ethanol 70% aqueous acetone
a

83 1 42 4 25 2 97 8

GAE gallic acid equivalent from FolinCiocalteu method

close to three shows that reaction between Fremys 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 Fremys 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 antioxidative index Ioxygen shows an almost linear response to the mate extract added to peroxidating lipid emulsion. The ethanol and methanol extracts are less efcient, while 70% aqueous acetone has the highest effect. The efciency of aqueous extract to suppress lipid oxidation was further conrmed in liposomes, where aqueous extract resulted in a signicant 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 signicant (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 protection (P < 0.0001) than rosemary in this specic 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 protected 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).

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Eur Food Res Technol Fig. 1 Reduction of ESR signal of Fremys salt (700 lmol) with the addition of mate aqueous extract

Table 2 Antioxidative index (Ioxygen) based on oxygen depletion rate in peroxidating methyl linolate emulsion for mate extracts obtained using different solvents Solvent Water Conc. (lg mate dried leaves mL1) 2.50 5.00 7.50 10.00 Fig. 2 Fremys salt reduced by increasing amount of aqueous extract of mate expressed in gallic acid equivalents. From linear regression the radical scavenging capacity of the extract is estimated to be 3.3 0.1 molecules of Fremys salt/molecules of GAE in extract Methanol Ethanol 70% aqueous acetone
a

Ia oxygen 0.49 0.03 0.37 0.08 0.30 0.03 0.17 0.01 0.19 0.03 0.22 0.03 0.10 0.03

10.00 10.00 10.00

Average of three determinations SD

In conclusion, mate is an attractive alternative for protection of pre-cooked meat products, as was demonstrated in this present study for pre-cooked chicken meat balls. The efcient protection may at least partially be mediated

through a synergistic interaction with vitamin E. Such synergistic effects could involve radical scavenging by vitamin E in the lipid phase followed by regeneration of vitamin E by water soluble phenolics from mate at the

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300 250 IP (min) 200 150 100 50 0 control 125 250 500 1.000 -1 Antioxidant concentration (mg dried leaves mL ) 3.000

Vit. E (ug alfa-tocoferol/g meat)

5,00 4,00 3,00 2,00 1,00 0,00 0 1 3 6 8 10

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

Days of storage

80 TBARS (umol MDA/kg meat) 70 60 50 40 30 20 10 0 0 1 3 6 Days of storage 8 10

Fig. 6 Effect of mate aqueous extracts (lled square, control; lled 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

interphase between lipids and the aqueous meat. Development of new chicken meat marketing will, however, depend on sensory products with mate added and such studies being conducted.

phase of the products for evaluation of are currently

Fig. 4 Formation of secondary lipid oxidation products in precooked chicken meat balls with and without addition of mate aqueous extractexperiment 1 (lled square, control; lled 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 malonaldialdehyde kg1 of meat). Mean values of two meat balls with two repetitions each

FORSK Acknowledgments This research was sponsored by the O Committee, as part of the research program FOODANTIOX-New Antioxidant Strategies for Food Quality and Consumer Health. The o Paulo Research Foundation) authors thank FAPESP (The State of Sa for nancial support such as travel and housing grant.

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1. Nissen LR, Maansson L, Bertelsen G, Huynh-Ba T, Skibsted LH (2000) J Agric Food Chem 48:55485556 2. Bragagnolo N, Danielsen B, Skibsted LH (2007) Innov Food Sci Technol 8:2427 Arce 3. Racanicci AMC, Danielsen B, Menten JFM, Regitano-d MAB, Skibsted LH (2004) Eur Food Res Technol 218:521524 pez P, Giberti G, Coussio J, Ferraro G (2001) Fito4. Filip R, Lo terapia 72:774778 5. Becker EM, Nissen LR, Skibsted LH (2004) Eur Food Res Technol 219:561571 6. Amerine MA, Ough CS (1980) In: Wiley J (ed) Methods for analysis of must and wines. Wiley-Interscience Publication, New York, pp 181184 7. Hu M, Skibsted LH (2002) Food Chem 76:327333 8. Roberts WG, Gordon MH (2003) J Agric Food Chem 51:1486 1493 9. Rdtjer A, Skibsted LH, Andersen ML (2006) Food Chem 99:6 14 10. Madsen HL, Srensen B, Skibsted LH, Bertelsen G (1998) Food Chem 63:173180 11. Jensen C, Guidera J, Skovgaard IM, Staun H, Skibsted LH, Jensen SK, Mller AJ, Buckley J, Bertelsen G (1997) Meat Sci 55:491500 12. Filip R, Ferrato GE (2003) Eur J Nutr 42:5054 13. Bragagnolo N, Danielsen B, Skibsted LH (2005) Eur Food Sci Technol 221:610615

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TBARS (umol MDA/kg meat)

80 70 60 50 40 30 20 10 0 0 1 3 6 8 10

Days of storage

Fig. 5 Formation of secondary lipid oxidation products in precooked chicken meat balls with and without addition of spices experiment 2 (lled square, control; lled triangle, rosemary 0.05% of dried leaves; open triangle, rosemary 0.10% of dried leaves; lled 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

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