Abstract

Rosemary (Rosmarinus officinalis) is known for their sensory characteristics and antioxidant properties, mainly due to the presence of several phenolic compounds. The aim of this work, was determine the antioxidant activity and apply the Rosemary lyophilized extract (RLE) in chicken burger, for assess their ability to reduce the lipid oxidation. Total antioxidant capacity and phenolic compounds profile were analyzed by colorimetric tests and liquid chromatography analysis, respectively. Thiobarbituric acid reactive substances assay was used to evaluate the ability of the RLE to prevent lipid peroxidation in chicken burger stored at 4 °C. Three treatments of chicken burgers were prepared (T1 – control, without addition of synthetic antioxidant BHT: butylated hydroxytoluene or RLE), T2 – with addition of BHT, and T3 – experimental, containing RLE). The high contents of total phenolic compounds (40.91 mg GAE g^-1: Gallic Acid Equivalent) and total flavonoids (24.26 mg QE g^-1: Quercetin Equivalents) were found in RLE. Rutin was the major phenolic compound identified in the RLE. The RLE showed strong antioxidant capacity and inhibited 48.29% of lipid oxidation (21 days of storage) in comparison to the control (T1), with low production of malonaldehyde, which has potential to be used in chicken burgers.

Keywords:
lipid oxidation; antioxidant assays; TBARS; HPLC; Rosmarinus officinalis

1 Introduction

Natural antioxidants offer alternatives approach to prevent the oxidative deterioration of foods. Such compounds found in plants have attracted considerable interest in preventing the auto-oxidation of fat and oil in foods (Fernández-Ginés et al., 2005Fernández-Ginés, J. M., Fernández-López, J., Sayas-Barberá, E., & Pérez-Alvarez, J. A. (2005). Meat products as functional foods: a review. Journal of Food Science, 70(2), R37-R43. http://dx.doi.org/10.1111/j.1365-2621.2005.tb07110.x.
http://dx.doi.org/10.1111/j.1365-2621.20... ; Mariutti et al., 2008Mariutti, L. R. B., Orlien, V., Bragagnolo, N., & Skibsted, L. H. (2008). Effect of sage and garlic on lipid oxidation in high-pressure processed chicken meat. European Food Research and Technology, 227(2), 337-344. http://dx.doi.org/10.1007/s00217-007-0726-5.
http://dx.doi.org/10.1007/s00217-007-072... ). Antioxidant activities of herbs, spices, vegetables and other extracts has been linked to phenolic compounds, suggesting that the antioxidant action is similar to that of the synthetic additives (Asolini et al., 2006Asolini, F. C., Tedesco, A. M., Carpes, S. T., Ferraz, C., & Alencar, S. M. (2006). Antioxidant and antibacterial activities of phenolic compounds from extracts of plants used as tea. Brazilian Journal of Food Technology, 9(3), 209-215.; Rêgo et al., 2011Rêgo, N. O. Jr., Fernandez, L. G., Castro, R. D., Silva, L. C., Gualberto, S. A., Pereira, M. L. A., & Silva, M. V. (2011). Bioactive compounds and antioxidant activity of crude extracts of caatinga plant species. Brazilian Journal of Food Technology, 14(1), 50-57.). Antioxidants act as scavengers of free radicals and oxygen, which reduce the progress of lipid peroxidation. However, it is need hinder the free radicals production at start of the reaction sequence or to interrupt the sequence at some point (Singleton et al., 1999Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods in Enzymology, 299, 152-178. http://dx.doi.org/10.1016/S0076-6879(99)99017-1.
http://dx.doi.org/10.1016/S0076-6879(99)... ).

Currently, efforts have increased to elucidate the effects of the natural antioxidants present in vegetables and food matrices (Rather et al., 2016Rather, S. A., Masoodi, F. A., Akhter, R., Rather, J. A., & Shiekh, K. A. (2016). Advances in use of natural antioxidants as food additives for improving the oxidative stability of meat products. Madridge Journal of Food Technology, 1(1), 10-17.; Reis et al., 2017Reis, A. S., Diedrich, C., Moura, C., Pereira, D., Almeida, J. F., Silva, L. D., Plata Oviedo, M. S. V., Tavares, R. A. W., & Carpes, S. T. (2017). Physico-chemical characteristics of microencapsulated propolis co-product extract and its effect on storage stability of burger meat during storage at -15 °C. Food Science and Technology, 76, 306-313.). The bioactivity of phenolic compounds depends largely of the number of hydroxyl groups linked to the aromatic ring and their relative orientation in the molecular structure. Different assays have been used to investigate the antioxidant activity of plants extracts in several matrices such as foods, natural products and body fluids (Erkan et al., 2008Erkan, N., Ayranci, G., & Ayranci, E. (2008). Antioxidant activities of Rosemary (Rosmarinus officinalis L.) extract, black seed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chemistry, 110(1), 76-82. PMid:26050168. http://dx.doi.org/10.1016/j.foodchem.2008.01.058.
http://dx.doi.org/10.1016/j.foodchem.200... ; Reşat et al., 2013Reşat, A., Shela, G., Volker, B., Karen, M. S., Mustafa, O., & Kubilay, G. (2013). Methods of measurement and evaluation of natural antioxidant capacity activity (IUPAC Technical Report). Pure and Applied Chemistry, 85(5), 957-998.; Guo et al., 2016Guo, Q., Gao, S., Sun, Y., Gao, Y., Wang, X., & Zhang, Z. (2016). Antioxidant efficacy of Rosemary ethanol extract in palm oil during frying and accelerated storage. Industrial Crops and Products, 94(1), 82-88. http://dx.doi.org/10.1016/j.indcrop.2016.08.032.
http://dx.doi.org/10.1016/j.indcrop.2016... ).

The Rosemary belongs to the Lamiaceae and possesses a number of desirable technological properties. Most of the data found in the literature refers to the antioxidant, antimicrobial and anti-inflammatory potential of Rosemary, which is often related to the presence of rosmarinic acid and carnasol, among other polyphenols (Erkan et al., 2008Erkan, N., Ayranci, G., & Ayranci, E. (2008). Antioxidant activities of Rosemary (Rosmarinus officinalis L.) extract, black seed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chemistry, 110(1), 76-82. PMid:26050168. http://dx.doi.org/10.1016/j.foodchem.2008.01.058.
http://dx.doi.org/10.1016/j.foodchem.200... ; Genena et al., 2008Genena, A. K., Hense, H., Smania, A. Jr, & Souza, S. M. (2008). Rosemary (Rosmarinus officinalis): composition study, antioxidant and antimicrobial activities of extracts obtained with supercritical carbon dioxide. Food Science and Technology, 28(2), 463-469. http://dx.doi.org/10.1590/S0101-20612008000200030.
http://dx.doi.org/10.1590/S0101-20612008... ; Pour et al., 2014Pour, A. R., Mirzargar, S. S., Soltani, M., Mousavi, H. A. E., & Seyed, A. M. (2014). The antibacterial effects of Cuminum cyminum L. and Rosmarinus officinalis extracts and essential oil against Lactococcus garvieae in laboratory conditions on rainbow trout. European Journal of Experimental Biology, 4(1), 456-463.). Rosemary extracts prevent autooxidation in sunflower oil (Chen et al., 2014Chen, X., Zhang, Y., Zu, Y., Yang, L., Lu, Q., & Wang, W. (2014). Antioxidant effects of Rosemary extracts on sunflower oil compared with synthetic antioxidants. International Journal of Food Science & Technology, 49(2), 385-391. http://dx.doi.org/10.1111/ijfs.12311.
http://dx.doi.org/10.1111/ijfs.12311... ) and it is used as natural antioxidant to inhibit lipid oxidation in pork-based products (Lara et al., 2011Lara, M. S., Gutierrez, J. I., Timón, M., & Andrés, A. I. (2011). Evaluation of two natural extract (Rosemarinus officinalis L. and Melissa officinales L.) as antioxidants in cooked pork patties packed in MAP. Meat Science, 88(3), 481-488. PMid:21345605. http://dx.doi.org/10.1016/j.meatsci.2011.01.030.
http://dx.doi.org/10.1016/j.meatsci.2011... ; Hać-Szymańczuk et al., 2011Hać-Szymańczuk, E., Lipińska, E., & Stasiuk, M. (2011). The effect of Rosemary preparations on the microbial quality and TBARS value of model pork batters. Acta Science Polonorum: Technologia Alimentaria, 10(2), 165-174.), and other types of foods such as sausages (Georgantelis et al., 2007Georgantelis, D., Ambrosiadis, I., Katikou, P., Blekas, G., & Georgakis, S. A. (2007). Effect of Rosemary extract, chitosan and α-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4 °C. Meat Science, 76(1), 172-181. PMid:22064204. http://dx.doi.org/10.1016/j.meatsci.2006.10.026.
http://dx.doi.org/10.1016/j.meatsci.2006... ), and chicken nuggets (Teruel et al., 2015Teruel, M. R., Garrido, M. D., Espinosa, M. C., & Linares, M. B. (2015). Effect of different format-solvent Rosemary extracts (Rosmarinus officinalis) on frozen chicken nuggets quality. Food Chemistry, 172(1), 40-46. PMid:25442521. http://dx.doi.org/10.1016/j.foodchem.2014.09.018.
http://dx.doi.org/10.1016/j.foodchem.201... ).

Lipid oxidation in foods is one of the main factors that affect the product quality such as, flavour, colour and texture, reducing the nutritional value. Oxidative processes in meat products are influenced by polyunsaturated fatty acids in the presence of iron, presence of prooxidants and endogenous antioxidants, action of oxygen, the addition of salt and by mechanical processes (Min & Ahn, 2005Min, B., & Ahn, D. U. (2005). Mechanism of lipid peroxidation in meat and meat products: a review. Food Science and Biotechnology, 14(1), 152-153.). According to Devatkal & Naveena (2010)Devatkal, S. K., & Naveena, B. M. (2010). Effect of salt, kinnow and pomegranate fruit by-product powders on color and oxidative stability of raw ground goat meat during refrigerated storage. Meat Science, 85(2), 306-311. PMid:20374904. http://dx.doi.org/10.1016/j.meatsci.2010.01.019.
http://dx.doi.org/10.1016/j.meatsci.2010... lipid oxidation is a chain reaction so that polyunsaturated fatty acids react sequentially with oxygen molecules by free radical mechanism making peroxides. The addition of antioxidants in meat products are used to prolong the shelf life, increase safety and prevent damage caused by lipid oxidation (Devatkal & Naveena, 2010Devatkal, S. K., & Naveena, B. M. (2010). Effect of salt, kinnow and pomegranate fruit by-product powders on color and oxidative stability of raw ground goat meat during refrigerated storage. Meat Science, 85(2), 306-311. PMid:20374904. http://dx.doi.org/10.1016/j.meatsci.2010.01.019.
http://dx.doi.org/10.1016/j.meatsci.2010... ; Chen et al., 2014Chen, X., Zhang, Y., Zu, Y., Yang, L., Lu, Q., & Wang, W. (2014). Antioxidant effects of Rosemary extracts on sunflower oil compared with synthetic antioxidants. International Journal of Food Science & Technology, 49(2), 385-391. http://dx.doi.org/10.1111/ijfs.12311.
http://dx.doi.org/10.1111/ijfs.12311... ).

Synthetic phenolic preservatives such as tertiary-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoleune (BHT), and propyl gallate (PG) are approved for use in meat processing in many countries. However, concern about the health risks associated with chronic consumption of these chemical substances are getting attention in the search for natural substances with antioxidant activity (Ibrahim et al., 2010Ibrahim, H. M., Abou-Arab, A. A., & Abu Salem, F. M. (2010). Addition of some natural plant extracts and their effects on lamb patties quality. Journal of Food Technology, 8(3), 134-142. http://dx.doi.org/10.3923/jftech.2010.134.142.
http://dx.doi.org/10.3923/jftech.2010.13... ; Shahidi & Zhong, 2010Shahidi, F., & Zhong, Y. (2010). Novel antioxidants in food quality preservation and health promotion. European Journal of Lipid Science and Technology, 112(9), 930-940. http://dx.doi.org/10.1002/ejlt.201000044.
http://dx.doi.org/10.1002/ejlt.201000044... ; Rather et al., 2016Rather, S. A., Masoodi, F. A., Akhter, R., Rather, J. A., & Shiekh, K. A. (2016). Advances in use of natural antioxidants as food additives for improving the oxidative stability of meat products. Madridge Journal of Food Technology, 1(1), 10-17.).

The aim of this work was to determine the antioxidant properties and phenolic profile by liquid chromatography, and the effects of adding of Rosemary lyophilized extract (RLE) in chicken burgers, in order to avoid the lipid oxidation during cold storage at 4 °C.

2 Materials and methods

2.1 Chemicals

Gallic acid, ferulic acid, caffeic acid, p-coumaric acid, trans-cinnamic acid, kaempferol, rutin, and quercetin were obtained from Sigma–Aldrich (Hamburg, Germany). DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS^•+ (2,2’-azino-di(3-ethylbenzthiazoline-6-sulphonic acid), TPTZ (2,4,6-Tri(2-pyridyl)-s-triazine), trichloroacetic acid (TCA), Folin–Ciocalteu phenol reagents, Trolox, linoleic acid, β-carotene, and Tween 40 were obtained from Sigma Aldrich (Sternheim, Germany). Ethylenediaminetetraacetic acid (EDTA), thiobarbituric acid (TBA), chloroform, ethyl alcohol and methanol were purchased from Vetec (Sao Paulo, Brazil).

2.2 Preparation of Rosemary lyophilized extract (RLE)

The preparation and the analysis of RLE were described by Carpes et al. (2008)Carpes, S. T., Prado, A., Moreno, I. A. M., Mourão, G. B., Alencar, S. M., & Masson, M. L. (2008). Screening of the antioxidant potential of bee pollen produced in the southern region of Brazil. Química Nova, 31(7), 1660-1664. http://dx.doi.org/10.1590/S0100-40422008000700011.
http://dx.doi.org/10.1590/S0100-40422008... . The Rosmarinus officinalis samples were cultivated in Pato Branco, Paraná, Brazil. The leaves were collected, dried in a air circulation oven at 35 °C and ground in analytical mill. The leaves (2 g) were incubated with 15 mL of 80% ethanol (v/v), at 70 °C in water bath for 30 min. Afterwards, the extract was filtered and stored at –5 °C. The supernatant was lyophilized (Liotop L101, São Carlos, Brazil). The Rosemary lyophilized extract was named as RLE and used in total phenols, flavonoids, antioxidant activity, and HPLC analysis. The extraction was performed in triplicate.

2.3 Total phenols and total flavonoids

Total phenolic compounds were quantified by Folin-Ciocalteu method described by Singleton et al. (1999)Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau reagent. Methods in Enzymology, 299, 152-178. http://dx.doi.org/10.1016/S0076-6879(99)99017-1.
http://dx.doi.org/10.1016/S0076-6879(99)... , using gallic acid as the standard. The results were expressed as mg GAE g^-1 of sample (GAE: Gallic Acid Equivalent). The content of total flavonoids was quantified according to Carpes et al. (2008)Carpes, S. T., Prado, A., Moreno, I. A. M., Mourão, G. B., Alencar, S. M., & Masson, M. L. (2008). Screening of the antioxidant potential of bee pollen produced in the southern region of Brazil. Química Nova, 31(7), 1660-1664. http://dx.doi.org/10.1590/S0100-40422008000700011.
http://dx.doi.org/10.1590/S0100-40422008... . Results were interpolated with quercetin calibration curve and presented in mg QE g^-1 of sample (QE: Quercetin Equivalent).

2.4 HPLC-DAD-UV-Vis profile

The analysis of phenolic compounds in RLE was carried out using a Dionex Ultimate 3000 chromatograph equipped with an Acclaim® 120 C18 colunn at 40 °C with a flow rate of 1.0 mL min^-1 and a photodiode array detector (HPLC/PDA). The mobile phases were carried out according to Rodrigues et al. (2015)Rodrigues, C. V., Silva, M. V., Santos, A. R., Zielinski, A. A. F., & Haminiuk, C. W. I. (2015). Evaluation of hot and cold extraction of bioactive compounds in teas. International Journal of Food Science & Technology, 50(9), 2038-2045. http://dx.doi.org/10.1111/ijfs.12858.
http://dx.doi.org/10.1111/ijfs.12858... and Chromeleon software was used in this experiment. Extracts were filtered through a 0.22 µm filter nylon syringe (Millipore, São Paulo, Brazil). Ferulic acid, gallic acid, p-coumaric acid, trans-cinnamic acid, kaempferol, rutin, and quercetin were examined. The assays were carried out in triplicate.

2.5 Antioxidant capacity tests

DPPH radical assay

The DPPH free radical scavenging activity was realized according Brand-Williams et al. (1995)Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft + Technologie, 28(1), 25-30. http://dx.doi.org/10.1016/S0023-6438(95)80008-5.
http://dx.doi.org/10.1016/S0023-6438(95)... . 0.5 mL of RLE 80 g/L, 3.0 mL of ethanol and 0.3 mL of 0.5 mM DPPH solution in ethanol were mixed. The absorbance was measured using a spectrophotometer (Bel Photonics 2000, Piracicaba, Brazil) at 517 nm. The concentration required to obtain a 50% antioxidant effect value (EC50) was calculated by linear regression of graphs where the axis of the abscissas is the concentration in mg/mL and the ordinal axis is the antioxidant activity (%). The assays were carried out in triplicate. Standard solutions of BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole) and alfa-tocopherol were used at final concentrations of 0.1 mg mL^–1, as positive controls.

ABTS^•+ scavenging activity

The ABTS assay was performed according to Re et al. (1999)Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS•+ radical cation decolorization assay. Free Radical Biology & Medicine, 26(9-10), 1231-1237. PMid:10381194. http://dx.doi.org/10.1016/S0891-5849(98)00315-3.
http://dx.doi.org/10.1016/S0891-5849(98)... . ABTS solution (1 mL) was diluted with ethanol (60 mL) until the absorbance 0.70 at 734 nm in spectrophotometer (UV-VIS Bel Photonics 2000 Piracicaba, Brazil). Trolox was used as reference and the result was expressed as mmol/g of TEAC (Trolox-equivalent antioxidant capacity). All assays were made in triplicate.

Coupled oxidation of β-carotene/linoleic acid assay

The β-carotene bleaching assay was determined according to Ahn et al. (2004)Ahn, M. R., Kumazawa, S., Hamasaka, T., Bang, K.-S., & Nakayama, T. (2004). Antioxidant activity and constituents of propolis collected in various areas of Korea. Journal of Agricultural and Food Chemistry, 52(24), 7286-7292. PMid:15563208. http://dx.doi.org/10.1021/jf048726s.
http://dx.doi.org/10.1021/jf048726s... . About 10 mg of β-carotene, 40 mg of linoleic acid and 400 mg Tween 40 were measured, and dissolved in 100 mL of chloroform. An aliquot of this emulsion (3 mL) and 300 μL of the RLE (6.67 mg mL^-1) was mixed and absorbance read immediately at 470 nm, and at 20 min intervals for 2 hours incubation at 50 °C. Antioxidant activity was presented as percent of inhibition. The BHT, BHA e α-tocopherol were evaluated in the final concentration of 0.1 mg mL^-1 at 470 nm. The assays were effectuated in triplicate.

Ferric reducing antioxidant potential (FRAP) assay

The FRAP assay was performed as described by Pulido et al. (2000)Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry, 48(8), 3396-3402. PMid:10956123. http://dx.doi.org/10.1021/jf9913458.
http://dx.doi.org/10.1021/jf9913458... . 25 mL of 0.3 M acetate buffer, 2.5 mL of 10 mM TPTZ solution, and 2.5 mL of 20 mM ferric chloride were used to obtain the reagent. 3 mL of FRAP reagent freshly was added to 100 µL of the extract and incubated in a water bath for 30 min at 37 °C. FeSO₄7H₂O standard curve was prepared and absorbance was measured at 595 nm. FRAP values were expressed as µmol Fe²⁺ g^-1 of sample. The assays were made in triplicate.

2.6 Preparation of the chicken burgers

The meat used for the formulations of the chicken burgers was obtained in the city of Pato Branco – PR, Brazil. The following formulation as used: 6 kg of chicken meat containing 10% breast meat, 55% thigh meat and drumstick and 15% of fat from chicken skin. The meat was minced and homogenized with iced water (13.8%), hydrated textured soy protein (0.9%), isolated soy protein (1%), salt (1.4%), curing salts (0.5%), ground white pepper (0.2%), garlic powder (0.1%), aroma of onion (0.1%) and cassava starch (2%). This basic formulation was divided into 3 lots. The first one was designated as the control without antioxidant addition (T1). The second lot was prepared by adding the BHT (butylated hydroxytoluene) at a concentration of 0.01% (T2). The third lot received Rosemary lyophilized extract at a concentration of 0.02% (T3). After the complete homogenization, the chicken burgers at a mean weight of 100 g were molded by hand press within the mold plate. After this, the burgers were packed in individual polyethylene plastic and stored at 4 °C.

2.7 Oxidative stability (TBARS)

The methodology of Raharjo & Sofos (1993)Raharjo, S., & Sofos, J. N. (1993). Methodology for measuring malonaldehyde as a product of lipid peroxidation in muscle tissues: A review. Meat Science, 35(2), 145-169. PMid:22061027. http://dx.doi.org/10.1016/0309-1740(93)90046-K.
http://dx.doi.org/10.1016/0309-1740(93)9... was used to evaluate oxidative stability of the chicken burgers formulations. Measurements were carried out on the day of the chicken burgers production (0) and after 7, 14 and 21 days of storage at 4 °C. For evaluation of aldehydes, 5 g of sample in 25 mL of TCA diluted with EDTA were homogenized in ultra Turrax (MA-102 Marconi, Piracicaba, Brazil). After filtration, the supernatant was reacted with a solution of TBA (thiobarbituric acid) at 95 °C for 40 min in water bath, forming a coloured complex. Substances reacting with thiobarbituric acid were measured spectrophotometrically at 532 nm. TBARS levels were expressed as mg of malonaldehyde kg^-1 sample. Lipid oxidation analysis was done in triplicate.

3 Results and discussion

3.1 Total phenols, total flavonoids and preliminary chromatographic analysis

Among the most abundant metabolites in vegetables (also known as secondary metabolites) are flavonoids and phenolic acids, which are known by antioxidant activity (Reşat et al., 2013Reşat, A., Shela, G., Volker, B., Karen, M. S., Mustafa, O., & Kubilay, G. (2013). Methods of measurement and evaluation of natural antioxidant capacity activity (IUPAC Technical Report). Pure and Applied Chemistry, 85(5), 957-998.; Dudonné et al., 2009Dudonné, S., Vitrac, X., Coutière, P., Woillez, M., & Mérillon, J.-M. (2009). Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS•+, FRAP, SOD, and ORAC assays. Journal of Agricultural and Food Chemistry, 57(5), 1768-1774. PMid:19199445. http://dx.doi.org/10.1021/jf803011r.
http://dx.doi.org/10.1021/jf803011r... ). A positive correlation between total phenols and antioxidant activity of plant extracts has been reported (Abdel-Gawad et al., 2014Abdel-Gawad, M., El-Sayed, M., El-Wakil, E., & Abdel-Lateef, E. (2014). In vitro antioxidant, total phenolic and flavonoid contents of six allium species growing in Egypt. Journal of Microbiology, Biotechnology and Food Science, 3(4), 343-346.). The mean value of total phenols compounds determined in ethanolic extract of Rosemary using Folin Ciocalteu method was 40.15 ± 0.15 mg GAE g^-1 of sample and the total flavonoids content was 22.43 mg QE g^-1 of Rosemary.

The results of this study differ from those of Zheng and Wang (2001)Zheng, W., & Wang, S. Y. (2001). Antioxidant activity and phenolic compounds in selected herbs. Journal of Agricultural and Food Chemistry, 49(11), 5165-5170. PMid:11714298. http://dx.doi.org/10.1021/jf010697n.
http://dx.doi.org/10.1021/jf010697n... , where 39 aromatic plants from China were evaluated; among them, the Rosemary was extracted with 15 mL of 75 mM phosphate buffer (pH 7.0) and it was found 2.19 ± 0.15 mg of GAE g^-1 of dry matter. Afonso et al. (2013)Afonso, M. S., Silva, A. M. O., Carvalho, E. B., Rivelli, D. P., Barros, S. B., Rogero, M. M., Lottenberg, A. M., Torres, R. P., & Mancini-Filho, J. (2013). Phenolic compounds from Rosemary (Rosmarinus officinalis L.) attenuate oxidative stress and reduce blood cholesterol concentrations in diet-induced hypercholesterolemic rats. Nutrition & Metabolism, 10(1), 19. PMid:23374457. http://dx.doi.org/10.1186/1743-7075-10-19.
http://dx.doi.org/10.1186/1743-7075-10-1... evaluated the inhibition of oxidative stress in cells of mice fed with hypercholesterolemic diet enriched with aqueous extract of Rosemary and reported 16.67 ± 0.40 mg GAE g^-1 of Rosemary.

Mata et al. (2007)Mata, A. T., Proença, C., Ferreira, A. R., Serralheiro, M. L. M., Nogueira, J. M. F., & Araújo, M. E. M. (2007). Antioxidant and antiacetylcholinesterase activities of five plants used as Portuguese food species. Food Chemistry, 103(3), 778-786. http://dx.doi.org/10.1016/j.foodchem.2006.09.017.
http://dx.doi.org/10.1016/j.foodchem.200... , obtained values for the phenolic compounds in aqueous and ethanolic extracts of Rosemary varying from 58.4 to 73.5 mg of GAE g^-1, respectively. Justo et al. (2008)Justo, O. R., Moraes, Â. M., Barreto, G. P. M., Mercadante, A. Z., & Rosa, P. T. V. (2008). Evaluation of the antioxidant potential of plant extracts obtained by supercritical fluid extraction. Nature Chemistry, 31(7), 1699-1705. studying ethanolic extracts of Rosemary, obtained by supercritical extraction, found values from 55 to 65 mg GAE g^-1 (dry matter). These differences are probably due to differences in growth conditions. It is known that environmental differences (geographical and climatic) agronomical practices and post-harvest conditions have effect on the concentration of total phenols (Scalzo et al., 2005Scalzo, J., Politi, A., Pellegrini, N., Mezzetti, B., & Battino, M. (2005). Plant genotype affects total antioxidant capacity and phenolic contents in fruit. Nutrition, 21(2), 207-213. PMid:15723750. http://dx.doi.org/10.1016/j.nut.2004.03.025.
http://dx.doi.org/10.1016/j.nut.2004.03.... ; Hernanz et al., 2007Hernanz, D., Recamales, A. F., Meléndez-Martínez, A. J., Gonzalezmiret, M. L., & Heredia, F. J. (2007). Assessment of the differences in the phenolic composition of five strawberry cultivars (Fragaria x ananassa Duch.) grown in two different soilless systems. Journal of Agricultural and Food Chemistry, 55(5), 1846-1852. PMid:17279770. http://dx.doi.org/10.1021/jf063189s.
http://dx.doi.org/10.1021/jf063189s... ; Correia et al., 2016Correia, S., Gonçalves, B., Aires, A., Silva, A., Ferreira, L., Carvalho, R., Fernandes, H., Freitas, C., Carnide, V., & Silva, A. P. (2016). Effect of harvest year and altitud on nutritional and biometric characteristics of blueberry cultivars. Journal of Chemistry, 1(1), 1-12. http://dx.doi.org/10.1155/2016/8648609.
http://dx.doi.org/10.1155/2016/8648609... ).

In this study, three flavonoids (kaempferol, rutin, quercetin) and four phenolic acids (gallic acid, p-coumaric acid, p-cinnamic acid, ferulic acid) were identified and quantified by liquid chromatography (Table 1). Among the individuals phenols identified, the rutin showed the higher concentration (33.06 ± 0.5 mg 100 g^-1). In addition, these substances when present in natural extracts added in food products, such as meat derived-products, are allegedly responsible to avoid the lipid oxidation (Reis et al., 2017Reis, A. S., Diedrich, C., Moura, C., Pereira, D., Almeida, J. F., Silva, L. D., Plata Oviedo, M. S. V., Tavares, R. A. W., & Carpes, S. T. (2017). Physico-chemical characteristics of microencapsulated propolis co-product extract and its effect on storage stability of burger meat during storage at -15 °C. Food Science and Technology, 76, 306-313.).

Wojdylo et al. (2007)Wojdylo, A., Oszmianski, J., & Czemerys, R. (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Journal of Food Chemistry, 105(3), 940-949. http://dx.doi.org/10.1016/j.foodchem.2007.04.038.
http://dx.doi.org/10.1016/j.foodchem.200... analysed 32 species of aromatic herbs from Poland, five of them from the family Labiatae; they analysed the phenolic profile of Rosemary extracts and the results support the findings of this study. According to authors Pérez-Fons et al. (2010)Pérez-Fons, L., Garzón, M. T., & Micol, V. (2010). Relationship between the antioxidant capacity and effect of Rosemary (Rosmarinus officinalis L.) polyphenols on membrane phospholipid order. Journal of Agricultural and Food Chemistry, 58(1), 161-171. PMid:19924866. http://dx.doi.org/10.1021/jf9026487.
http://dx.doi.org/10.1021/jf9026487... and Wojdylo et al. (2007)Wojdylo, A., Oszmianski, J., & Czemerys, R. (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Journal of Food Chemistry, 105(3), 940-949. http://dx.doi.org/10.1016/j.foodchem.2007.04.038.
http://dx.doi.org/10.1016/j.foodchem.200... the antioxidant activity of ethanolic extracts of Rosemary is mainly due to phenolic compounds (phenolic acids and flavonoids).

3.2 Antioxidant activities

The antioxidant capacity of RLE determined by distinct in-vitro methods as DPPH, ABTS, FRAP and β-carotene/linoleic acid (Table 2). Ethanolic extract of Rosemary had lower antioxidant activities than the commercial antioxidants by the DPPH method (EC₅₀ = 127.33 ± 0.12 µg/mL) and by the method of scavenging of the radical ABTS^•+ (203.11 ± 1.10 µmol g^-1 of Trolox). Alpha-tocopherol showed the highest antioxidant activity in the DPPH^• and ABTS^•+ tests.

The EC₅₀ values of RLE were very low (127.33 µg/mL), indicating a high antioxidant activity in Rosemary (Table 2). Erkan et al. (2008)Erkan, N., Ayranci, G., & Ayranci, E. (2008). Antioxidant activities of Rosemary (Rosmarinus officinalis L.) extract, black seed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chemistry, 110(1), 76-82. PMid:26050168. http://dx.doi.org/10.1016/j.foodchem.2008.01.058.
http://dx.doi.org/10.1016/j.foodchem.200... that reported a EC₅₀ value of 54.0 μg mL^-1 in Rosemary methanolic extracts using a Soxhlet apparatus, i.e, a lower than those found in this study (Table 2). Rodríguez-Rojo et al. (2012)Rodríguez-Rojo, S., Visentin, A., Maestri, D., & Cocero, M. J. (2012). Assisted extraction of Rosemary antioxidants with green solvents. Journal of Food Engineering, 109(1), 98-103. http://dx.doi.org/10.1016/j.jfoodeng.2011.09.029.
http://dx.doi.org/10.1016/j.jfoodeng.201... studied Rosemary leaves collected in Spain (DPPH) and obtained values of EC₅₀ of 45 and 17 µg mL^-1 for ethanolic and aqueous extraction, respectively. It is suggested that the choice of solvent must be primarily related to the use of the extract; in the case of an aqueous extract of Rosemary, which is rich in rosmarinic acid, would be effective as an antioxidant in hydrophilic systems, while in lipophilic systems, ethanolic extracts would be more appropriate, owing to their higher content of carnosic acid. This refers that physiochemical nature of the phenolic compounds in the extracts may be more important for the antioxidant activity than the total phenols.

Justo et al. (2008)Justo, O. R., Moraes, Â. M., Barreto, G. P. M., Mercadante, A. Z., & Rosa, P. T. V. (2008). Evaluation of the antioxidant potential of plant extracts obtained by supercritical fluid extraction. Nature Chemistry, 31(7), 1699-1705. investigated the antioxidant properties of ethanolic extracts of Rosemary by the DPPH^• (EC₅₀) and ABTS^•+ methods, and found values of EC₅₀ varying from 120 to 9 µg mL^-1 and values of 190.96 ± 13.35 μM of Trolox g^-1 to 231.51 ± 11.90 μM of Trolox g^-1 by ABTS^•+ method.

The β-carotene is oxidized during β-carotene bleaching assay, and as result of this, smaller molecules are broken, the system loses its color (Carpes et al., 2008Carpes, S. T., Prado, A., Moreno, I. A. M., Mourão, G. B., Alencar, S. M., & Masson, M. L. (2008). Screening of the antioxidant potential of bee pollen produced in the southern region of Brazil. Química Nova, 31(7), 1660-1664. http://dx.doi.org/10.1590/S0100-40422008000700011.
http://dx.doi.org/10.1590/S0100-40422008... ) and the yellowish discolouration of the β-carotene can be measured spectrophotometrically. Rosemary lyophilized extract showed 92.31% of inhibition by the β-carotene method (extract concentration of 6.67 mg g^-1). When comparing the results of the RLE with the positive controls (BHA, BHT and alpha-tocopherol) by Tukey test, it can be seen that the RLE showed the lowest antioxidant capacity (Table 2). On the other hand, in the FRAP method, the RLE presented an antioxidant activity of 347.70 ± 0.69 µmol Fe⁺² g^-1, while the commercial antioxidants (BHA and BHT) showed higher values. This difference can be explained by the purity of the commercial additives, differing from the complexity of the studied RLE. In addition, consumers have increasingly preferred products containing natural additives because of concerns about adverse health effects of synthetic antioxidant such as BHT and BHA (Rather et al., 2016Rather, S. A., Masoodi, F. A., Akhter, R., Rather, J. A., & Shiekh, K. A. (2016). Advances in use of natural antioxidants as food additives for improving the oxidative stability of meat products. Madridge Journal of Food Technology, 1(1), 10-17.; Reis et al., 2017Reis, A. S., Diedrich, C., Moura, C., Pereira, D., Almeida, J. F., Silva, L. D., Plata Oviedo, M. S. V., Tavares, R. A. W., & Carpes, S. T. (2017). Physico-chemical characteristics of microencapsulated propolis co-product extract and its effect on storage stability of burger meat during storage at -15 °C. Food Science and Technology, 76, 306-313.). These results of the antioxidant assays clearly indicate that Rosemary extract have strong antioxidant capacity and can be used as natural antioxidant to preserve foods (Justo et al., 2008Justo, O. R., Moraes, Â. M., Barreto, G. P. M., Mercadante, A. Z., & Rosa, P. T. V. (2008). Evaluation of the antioxidant potential of plant extracts obtained by supercritical fluid extraction. Nature Chemistry, 31(7), 1699-1705.; Guo et al., 2016Guo, Q., Gao, S., Sun, Y., Gao, Y., Wang, X., & Zhang, Z. (2016). Antioxidant efficacy of Rosemary ethanol extract in palm oil during frying and accelerated storage. Industrial Crops and Products, 94(1), 82-88. http://dx.doi.org/10.1016/j.indcrop.2016.08.032.
http://dx.doi.org/10.1016/j.indcrop.2016... ).

3.3 Effect of Rosemary lyophilized extract on oxidative stability in chicken burgers

The oxidative degradation of lipids is one of the main reactions that occur in food products and can be inhibited by the use of preservatives in meat and meat products (Georgantelis et al., 2007Georgantelis, D., Ambrosiadis, I., Katikou, P., Blekas, G., & Georgakis, S. A. (2007). Effect of Rosemary extract, chitosan and α-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4 °C. Meat Science, 76(1), 172-181. PMid:22064204. http://dx.doi.org/10.1016/j.meatsci.2006.10.026.
http://dx.doi.org/10.1016/j.meatsci.2006... ; Guyon et al., 2016Guyon, C., Meynier, A., & De Lamballerie, M. (2016). Protein and lipid oxidation in meat: a review with emphasis on high-pressure treatments. Trends in Food Science & Technology, 50(1), 131-143. http://dx.doi.org/10.1016/j.tifs.2016.01.026.
http://dx.doi.org/10.1016/j.tifs.2016.01... ). Lipids are subject to a series of reactions which may lead to change in their structure and can affect the nutritional quality of the food products (Shahidi & Zhong, 2010Shahidi, F., & Zhong, Y. (2010). Novel antioxidants in food quality preservation and health promotion. European Journal of Lipid Science and Technology, 112(9), 930-940. http://dx.doi.org/10.1002/ejlt.201000044.
http://dx.doi.org/10.1002/ejlt.201000044... ).

TBARS test (thiobarbituric acid reactive substances) evaluates the quality of food product and is the most appropriate test for evaluating rancidity due to its simplicity and efficiency (Raharjo & Sofos, 1993Raharjo, S., & Sofos, J. N. (1993). Methodology for measuring malonaldehyde as a product of lipid peroxidation in muscle tissues: A review. Meat Science, 35(2), 145-169. PMid:22061027. http://dx.doi.org/10.1016/0309-1740(93)90046-K.
http://dx.doi.org/10.1016/0309-1740(93)9... ). In the meat products the TBARS test is important during meat preparation (grinding and mixing) and cooking, which favour the formation of malonaldehyde, a by-product of the decomposition of hydroperoxides formed during the oxidation process (Selani et al., 2011Selani, M. M., Contreras-Castillo, C. J., Shirahigue, L. D., Gallo, C. R., Plata-Oviedo, M., & Montes-Villanueva, N. D. (2011). Wine industry residues extracts as natural antioxidants in raw and cooked chicken meat during frozen storage. Meat Science, 88(3), 397-403. PMid:21342750. http://dx.doi.org/10.1016/j.meatsci.2011.01.017.
http://dx.doi.org/10.1016/j.meatsci.2011... ). The results obtained in the evaluation of lipid oxidation by TBARS in the three treatments are shown in Table 3.

On the day of processing, there was no significant difference among control (T1) and the burgers containing synthetic antioxidant (T2) or RLE (T3) (p>0.05). After seven days, the samples treated with BHT and RLE showed lower concentrations of malonaldehyde in comparison with the control (Table 3).

It was possible to show that the levels of malonaldehyde increased over 7 days of storage in all cases; however, from 14 days, the TBARS values declined with storage time. According to Ninan et al. (2008)Ninan, G., Bindu, J., & Joseph, J. (2008). Frozen storage studies of mince based products developes from tilapia (Oreochromis mossambicus, Peter 1852). Fishery Technology, 45(1), 35-42. the decrease is probably due to the interaction of lipid oxidized products (as malonaldehyde) with proteins. However, as reviewed by Angelini (2010)Angelini, M. F. C. (2010). Desenvolvimento do produto de conveniência Quenelle de tilápia (Oreochromis niloticus) (Master’s dissertation). Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, 2010., another possibility for decrease of the TBARS values can be explained by the oxidation products formed not be detected by this methodology. At 21 days, the chicken burgers without antioxidants (T1) had the highest content of malonaldehyde and there was significant difference (p<0.05) between the burgers containing BHT and those containing RLE (Table 3).

Estévez et al. (2007)Estévez, M., Ramírez, R., Ventanas, S., & Cava, R. (2007). Sage and Rosemary essential oils versus BHT for the inhibition of lipid oxidative reactions in liver pâté. Food Science and Technology, 40(1), 58-65. found 0.1% sage essential oil was more effective in inhibiting the generation of malonaldehyde in liver pâté during 90 days at 4 °C than 0.02% BHT. Also, the addition of 0.05, 0.10 and 0.15% of Salvia officinalis in Chinese sausage had lower TBARS values compared to controls (Zhang et al., 2013Zhang, L., Lin, Y. H., Leng, X. J., Huang, M., & Zhou, G. H. (2013). Effect of sage (Salvia officinalis) on the oxidative stability of Chinese-style sausage during refrigerated storage. Meat Science, 95(2), 145-150. PMid:23739264. http://dx.doi.org/10.1016/j.meatsci.2013.05.005.
http://dx.doi.org/10.1016/j.meatsci.2013... ). According to Brannan (2008)Brannan, R. G. (2008). Effect of grape seed extract on physicochemical properties of ground, salted, chicken thigh meat during refrigerated storage at different relative humidity levels. Journal of Food Science, 73(1), C36-C40. PMid:18211347. http://dx.doi.org/10.1111/j.1750-3841.2007.00588.x.
http://dx.doi.org/10.1111/j.1750-3841.20... , the oxidative rancidity occurs during frozen storage of beef and chicken. The microbiological and enzymatic reactions can be inhibited by low freezing temperatures however, the lipid oxidation continues in a reduced rate.

According to Al-Kahtani et al. (1996)Al-Kahtani, H. A., Abu-Tarboush, H. M., Bajaber, A. S., Atia, M., Abou-Arab, A. A., & El-Mojaddidi, M. A. (1996). Chemical changes after irradiation and post-irradiation storage in tilapia and Spanish mackerel. Journal of Food Science, 61(4), 729-733. http://dx.doi.org/10.1111/j.1365-2621.1996.tb12191.x.
http://dx.doi.org/10.1111/j.1365-2621.19... and Selani et al. (2011)Selani, M. M., Contreras-Castillo, C. J., Shirahigue, L. D., Gallo, C. R., Plata-Oviedo, M., & Montes-Villanueva, N. D. (2011). Wine industry residues extracts as natural antioxidants in raw and cooked chicken meat during frozen storage. Meat Science, 88(3), 397-403. PMid:21342750. http://dx.doi.org/10.1016/j.meatsci.2011.01.017.
http://dx.doi.org/10.1016/j.meatsci.2011... meat products are considered in a good state for consumption if they have values of below 3 mg of malonaldehyde kg^-1. According to these findings, the sausage containing RLE would be considered in good condition after 21 days of storage (1.51 mg malonaldehyde/kg sample) (Table 3). Therefore, the concentrations of RLE used in chicken burger were sufficient to maintain oxidative stability.

When compared to the control after 21 days of storage, the presence of BHT showed a reduction in TBARS values of 47.26%. At the end of storage time (21 days), the chicken burger containing RLE inhibited 48.29% of lipid oxidation in comparison to the control chicken burger (Table 4). This demonstrates the effectiveness of the Rosemary as antioxidant to prevent lipid oxidation in chicken burger during storage.

Teruel et al. (2015)Teruel, M. R., Garrido, M. D., Espinosa, M. C., & Linares, M. B. (2015). Effect of different format-solvent Rosemary extracts (Rosmarinus officinalis) on frozen chicken nuggets quality. Food Chemistry, 172(1), 40-46. PMid:25442521. http://dx.doi.org/10.1016/j.foodchem.2014.09.018.
http://dx.doi.org/10.1016/j.foodchem.201... studied the bioactive compounds from Rosmarinus officinalis in chicken nuggets during nine months of the storage at -18 °C, and found TBARS values of 4.07 to 5.88 mg malonaldehyde/kg sample. The effects of using natural herb extracts in inhibiting lipid oxidation were also reported by Ibrahim et al. (2010)Ibrahim, H. M., Abou-Arab, A. A., & Abu Salem, F. M. (2010). Addition of some natural plant extracts and their effects on lamb patties quality. Journal of Food Technology, 8(3), 134-142. http://dx.doi.org/10.3923/jftech.2010.134.142.
http://dx.doi.org/10.3923/jftech.2010.13... who analysed burgers containing extracts of jojoba, jatropha, ginseng and ginger. The extracts showed lower TBARS values than the control, inhibiting lipid oxidation during storage, at where lowest value of TBARS was found in burgers with the addition of ginseng extract.

4 Conclusion

This study confirms that RLE had strong anti-oxidative effects in chicken burgers, probably due to high antioxidant activity of the phenolic compounds such as acid gallic, catechin, p-coumaric acid, ferulic acid, p-cinnamic acid, rutin, quercetin, and kaempferol. The high capacity antioxidant of these compounds, combined with storage in a refrigerated temperature, the RLE has potential to be used in chicken burgers. Finding natural antioxidant equivalent or better than commercial antioxidants is an important challenge for food technology, principally due to the adverse health effects caused by synthetic antioxidants. Thus, the RLE provide good alternatives for consumer demand for healthy meat products.

Acknowledgements

The authors thank the National Council for Scientific and Technological Development (Process Number 481758/2012-7) for financial support and the Araucaria Foundation for the scholarships.

References

Publication Dates

History