The application of natural antioxidants via brine injection protects Iberian cooked hams against lipid and protein oxidation

Meat Science 116 (2016) 253–259

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The application of natural antioxidants via brine injection protects Iberian cooked hams against lipid and protein oxidation Mónica Armenteros a, David Morcuende b, Jesús Ventanas a, Mario Estévez b,⁎ a b

SiPA, University of Extremadura, Av. de las Ciencias s/n, 10003 Cáceres, Spain IPROCAR Research Institute, University of Extremadura, Av. de las Ciencias s/n, 10003 Cáceres, Spain

a r t i c l e

i n f o

Article history: Received 2 March 2015 Received in revised form 15 February 2016 Accepted 16 February 2016 Available online 17 February 2016 Keywords: Spices Fruits Cooked hams Lipid oxidation Protein oxidation

a b s t r a c t In response to the increasing consumers' mistrust in synthetic additives, the meat industry is focused on searching sources of natural antioxidants. Two different sources of natural antioxidants i) a mixture of garlic, cinnamon, cloves and rosemary essential oils and ii) a Rosa canina L. extract, were compared with a commercial antioxidant additive (Artinox®) for their ability to control protein and lipid oxidation in cooked hams after a settling period of 30 days and at the end of a chilled storage (150 days). The mixture of essential oils was the most effective against lipid oxidation while R. canina L. extracts were the most effective in controlling protein carbonylation at day 150. Accordingly, the use of these antioxidants via brine injection is a successful strategy to enhance the oxidative stability of cooked hams without modifying their physicochemical properties. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Cooked ham is a delicatessen meat product highly appreciated among European consumers (Toldrá, Mora, & Flores, 2010). The type and volume of injected brine, the rate and length of tumbling and the cooking time and temperature are among the most relevant technological factors having an impact on the quality of the final product (Delahunty, McCord, O'Neill, & Morrissey, 1997). This complex processing system could affect the oxidative stability of muscle lipids and proteins, and hence, the nutritional and sensory properties of cooked hams (St. Angelo et al., 1987). The heating process disrupts the muscle cell structure, inactivates antioxidative enzymes and releases catalytic iron from myoglobin leading to an intense prooxidant environment in which both, lipids and proteins, can be affected (Estévez, 2011; Kanner, 1994). Additionally, prior to consumption, cooked ham is commonly chilled or frozen and during this period, the oxidizing action of air and light may alter the color, aroma and flavor of hams and hence, consumers' acceptability (Ahn, Grün, & Mustapha, 2007). A recent study (Utrera, Armenteros, Ventanas, Solano, & Estévez, 2012) reported the potential impact of protein oxidation on the color and texture of cooked hams elaborated from previously frozen raw material. In order to inhibit oxidative reactions and as a result, protect muscle foods against their unpleasant effects, additives and ingredients with antioxidant potential are commonly used in the meat industry. Concerns over the safety of synthetic compounds and consumer's interest ⁎ Corresponding author. E-mail address: [email protected] (M. Estévez).

http://dx.doi.org/10.1016/j.meatsci.2016.02.027 0309-1740/© 2016 Elsevier Ltd. All rights reserved.

in the so-called natural ingredients, has prompted scientists to search alternative natural antioxidants derived from natural sources such as fruits, vegetables, seeds and spices (Cando, Morcuende, Utrera, & Estévez, 2014; Ganhão, Morcuende & Estévez, 2010; RodríguezCarpena, Morcuende, & Estévez, 2012; Yoo, Lee, Lee, Moon, & Lee, 2008). The potential of spices as natural antioxidants has been extensively reported and generally the Labitae family and particularly rosemary (Rosmarinus officinalis), are well known for their antioxidant properties (Nissen, Byrne, Bertelsen, & Skibsted, 2004). The effectiveness of rosemary essential oils as antioxidants has been demonstrated in a large variety of meat products including refrigerated beef, frozen pork patties or frankfurters (Djenane, Sánchez-Escalante, Beltrán, & Roncalés, 2003; Estévez, Morcuende, & Cava, 2005; Estévez, Ramírez, Ventanas, & Cava, 2007; Estévez, Ventanas & Cava, 2007; McCarthy, Kerry, Kerry, Lynch, & Buckley, 2001). Other spices like cinnamon (Cinnamomum verum) or clove (Syzygium aromaticum) have been shown to decrease lipid oxidation as effectively as certain synthetic antioxidants in cooked meat products (Dwivedi, Vasavada, & Cornforth, 2006; Jayathilakan, Sharma, Radhakrishna, & Bawa, 2007; McCarthy et al., 2001). Clove has been used as a condiment and reported to be rich in hydrolysable tannins and eugenol, which is known to show strong antioxidant activity (Ito, Murakami, & Yoshino, 2005). Cinnamon is rich in essential oils (mainly cinnamaldehyde and eugenol) which possess antimicrobial properties and also cinnamic aldehydes which have potential antioxidant properties (Murcia et al., 2004). However, the characteristic aroma of these spices limits their use (Craig, 1999). Garlic (Allium sativum) has also been proposed as a source of natural antioxidants. Garlic extracts have been reported to inhibit lipid oxidation

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by acting as hydroxyl radical scavengers in several meat products like dry-fermented sausages (Aguirrezábal, Mateo, Domínguez, & Zumalacárregui, 2000; Yang, Yasaei, & Page, 1993). Moreover, a wide variety of wild Mediterranean fruits and berries are found in the Mediterranean forest with powerful antioxidant activity. For instance, rose hips (Rosa canina L.) are rich in phenolic compounds and ascorbic acid and have been recently reported to be effective enhancers of the oxidative stability of meat patties (Ganhão, Morcuende, et al., 2010). However, only few studies have attempted to elucidate the effectiveness of mixtures of natural antioxidants against lipid and protein oxidation (Ahn et al., 2007). Additionally, the technological challenge of inoculating such phytochemicals in intact meat products such as cooked hams needs to be approached. Accordingly, the aim of this work was to investigate the effects of a spice mixture (garlic, cinnamon, cloves and rosemary), and a phenolicrich extract from R. canina on the physico-chemical properties and lipid and protein oxidation in cooked hams after a settling period of 30 days and at the end of a chilled storage (150 days). These effects were compared with those displayed by a commercial natural antioxidant (Artinox®) based on a mixture of citrate and erythorbate. 2. Material and methods 2.1. Materials The raw material, left hind legs from pure Iberian breed × Duroc pigs were supplied by Consorcio de Jabugo, S.A. The antioxidant blend of essential oils of garlic, cinnamon, cloves and rosemary was obtained from Aditivos Industriales, S.A. (San José, Costa Rica), whereas the commercial antioxidant namely Artinox® consists of a combination of additives (sodium citrate: sodium erythorbate; 1:1 w/w) from Chemital S.A. (Terrasa, Barcelona, Spain). Fruits from rose hip (R. canina L.) were collected at full ripeness in the Cáceres region (Spain) and subsequently frozen at − 80 °C until used. Other chemicals and reagents were purchased from Merck (Merck, Darmastadt, Germany), Panreac (Panreac Química, S.A., Barcelona, Spain) and Sigma Chemicals (Sigma-Aldrich, Steinheim, Germany). 2.2. Preparation of rose hip extracts For the preparation of rose hip extracts, 30 g of sample including peel and pulp were cut into pieces while the seeds were carefully removed. The fruit was ground, dispensed in a falcon tube and homogenized with 10 volumes (w/v) of absolute ethanol. The homogenates were centrifuged at 2600 × g for 10 min at 6 °C. The supernatants were collected and the residue was re-extracted once more following the procedure previously described. The two supernatants were combined, evaporated using a rota-evaporator and redissolved using 250 g of distilled water. Water solutions from rose hips were prepared and stored under refrigeration until used for the manufacture of cooked hams (less than 24 h). No insoluble fragments or residues were observed in the water solutions.

2.4. Manufacture of cooked hams The above mentioned left hind legs were used as raw material for the manufacture of cooked hams. The frozen legs were thawed in a cold chamber by keeping overnight at +4 °C, similarly to the industrial process. The legs were deboned while subcutaneous and intermuscular fat, connective tissue and rind were removed. Hams were injected with brine to increase their weight by 21% and to obtain 0.3% pentasodium tripolyphosphate, 0.05% sodium ascorbate, 1.8% NaCl and 0.01% sodium nitrite in the injected hams. Cold brine (− 2 °C) was injected using a multineedle injector equipped with a spraying system at constant pressure (Movistick 60PC, Metalquimia, Girona, Spain). Hams were then placed in a vacuum tumbler (Thermomat 8X, Metalquimia, Girona, Spain) at +4 °C at a pressure of 200 mbar. The tumbling schedule was set for the hams to rotate a total of 2000 times at 14 rpm. After a 48 h maturation period, the hams were packed in bags (CN330, Sealed Air, Italy), molded in 7 L capacity stainless steel molds (INOX.SERIE 444, Inoxnisge, Barcelona, Spain), placed in an automatic steam oven (FDC Cookline, Metalquimia, Girona, Spain) and cooked to an internal temperature of + 66 °C using an external temperature of + 68 °C. Upon completion of the cooking procedure, cooked hams were vacuumpacked following the aforementioned procedure for fresh hind legs and kept for five months at +4 °C. Microbiological analyses (anaerobic, enterobacterias, and Listeria monocytogenes) guaranteed the safety of the products during the whole storage (data not shown). 2.5. Experimental setting Depending on the addition of different ingredients through the injected brine, hams were randomly divided into three groups (n = 10) and each group was assigned to one of the following three treatments: Treatment 1 (T1), treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2 (T2), treated with Artinox® powder (3 g per kg of meat), and Treatment 3 (T3), treated with the experimental rose hip extract (300 mL per kg of meat) as antioxidant. Prior to the injection, the different sources of antioxidants were dissolved in distilled water and mixed with the brine. Treatments 1 and 3 were equivalent in total phenolic content (≈500 mg GAE/100 g sample) as measured by the Folin–Ciocalteu method described by Singleton and Rossi (1965) and were selected in accordance to preliminary studies to guarantee protection against oxidation phenomena. The addition of Artinox® followed the producer recommendation. For comparative purposes, some data from a previous study (Utrera et al., 2012) was used as a Control (no added antioxidants) as the raw material and processing were identical to those applied to the present samples. The cooked hams (n = 10 per treatment) were randomly divided into two groups for sampling 1) after a settling period of 30 days (n = 5) and 2) at the end of the chilled storage (150 days; n = 5). At sampling times, semimembranosus muscles from cooked hams were manually removed and subjected to the physico-chemical and lipid and protein oxidation analyses. 2.6. Physico-chemical analyses

2.3. Animals Thirty pure Iberian breed × Duroc pigs were free-range reared and fed on commercial diet based on grains and soybeans following the traditional livestock farming for Iberian pigs. The animals were slaughtered at 154 kg live weight. Carcasses were produced and deboned right after slaughter (hot deboning). Thirty left hind legs were obtained and subsequently vacuum-packed by reducing pressure to 10 mbar and using HF100 in the upper film with 34.0 cm3/m2 permeability to O2 at 23 °C and 85% HR and HF200 in the lower film with 18.0 cm3/m2 permeability to O2 at 23 °C and 85% HR (Mobepack Company, Salamanca, Spain) Vacuum-packed legs were kept frozen at −20 °C until their manufactured (5 months).

2.6.1. Proximate composition, water activity and pH of cooked hams Moisture, total protein, heme iron, nitrite and chloride content were determined using AOAC methods (AOAC, 2000). The method of Bligh and Dyer (1959) was used for isolation of fat from each sample. The water activity (aw) was determined by using a Lab Master aw system (Scientific Solutions, Hamilton Drive Mentor, OH, USA) and the pH of the cooked hams was analyzed using a pH meter Crisom (Crison Instruments, S.A., Barcelona, Spain). 2.6.2. Fatty acid profile Fatty acid methyl esters (FAMEs) were prepared by acidic esterification in the presence of sulfuric acid, following the method of López-Bote,

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Rey, Sanz, Gray, and Buckley (1997). FAMEs were analyzed using a Hewlett Packard, HP-6890N gas chromatograph, equipped with an on-column injector and a flame ionization detector, using a polyethyleneglycol capillary column (Supelcowas-10, Supelco, Bellefonte, PA) (60 m × 0.32 mm i.d. × 0.25 μm film thickness). Gas chromatograph oven program temperature was as follows: the initial temperature, set at 190 °C, was increased 2 °C/min up to 235 °C. It was then kept 15 min at that temperature and thereafter increased again 6 °C/min up to 250 °C. It remained at that final temperature for 20 min. Injector and detector temperature were 250 °C. Carrier gas was helium at a flow rate of 0.8 mL/min. Individual FAMEs peaks were identified by comparison of their retention times with those of standards (Sigma, St. Louis, MO). Tridecanoic acid was used as internal standard. Results were expressed as percentage of total fatty acid methyl esters. 2.6.3. Total phenolic content (TPC) Total phenolic content (TPC) was determined following to the Folin– Ciocalteu method described by Singleton and Rossi (1965). TPC was estimated from as standard curve of gallic acid, and results were expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample. 2.6.4. Determination of protein carbonyls Protein oxidation, as measured by the total carbonyl content, was evaluated by derivatization with dinitrophenylhydrazine (DNPH) according to the method described by Oliver, Ahn, Moerman, Goldstein, and Stadtman (1987) with slight modifications (Ganhão, Morcuende, et al., 2010). Samples (1 g) were minced and then homogenized 1:10 (w/v) in 20 mM sodium phosphate buffer containing 0.6 M NaCl (pH 6.5) using an ultraturrax homogenizer for 30 s. Two equal aliquots of 0.2 mL were taken from the homogenates and dispensed in 2 mL Eppendorf tubes. Proteins were precipitated by cold 10% TCA (1 mL) and subsequent centrifugation for 5 min at 620 g. One pellet was treated with 1 mL 2 M HCl (protein concentration measurement) and the other with an equal volume of 0.2% (w/v) DNPH in 2 M HCl (carbonyl concentration measurement). Both samples were incubated for 1 h at room temperature. Afterwards, samples were precipitated by 10% TCA (1 mL) and washed twice with 1 mL ethanol:ethyl acetate (1:1, v/v) to remove excess of DNPH. The pellets were then dissolved in 1.5 mL of 20 mM sodium phosphate buffer containing 6 M guanidine HCl (pH 6.5), stirred and centrifuges for 2 min at 620 g to remove insoluble fragments. Protein concentration was calculated from absorption at 280 nm using BSA as standard. The amount of carbonyls was expressed as nmol of carbonyl per mg of protein using an absorption coefficient of 21.0 nM−1 cm−1 at 370 nm for protein hydrazones. 2.6.5. Determination of TBARS numbers Thiobarbituric acid-reactive substances (TBARS) were determined using the method of Ganhão, Estévez & Morcuende (2011). Briefly, 5 g of cooked ham were dispensed in cone plastic tubes and homogenized with 15 mL perchloric acid (3.86%) and 0.5 mL BHT (4.2% in ethanol). The plastic tubes were immersed in an ice bath to minimize the development of oxidative reactions during extraction of TBARS. The slurry was filtered and centrifuged (600 × g for 4 min) and 2 mL aliquots were mixed with 2 mL thiobarbituric acid (0.02 M) in test tubes. The test tubes were placed in a boiling water bath (100 °C) for 45 min together with the tubes from the standard curve. After cooling, the absorbance was measured at 532 nm. The standard curve was prepared using a 1,1,3,3-tetraethoxypropane (TEP) solution (0.2268 g in 3.86% perchloric acid). The results were expressed as mg malondialdehyde (MDA) per kg of sample. 2.6.6. Color measurements Surface color measurement of semimembranosus muscle were performed using a Minolta chromameter CR-300 (Minolta Camera Corp.,

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Meter Division, Ramsey, NJ) with illuminant D65 and 0° standard observer. Before each measurement the chromameter was calibrated on the CIE color space system using a white tile. Three color indices were obtained: L* (lightness), a* (redness) and b* (yellowness) values. Saturation index (chroma) and Hue angle (h°) values were obtained by the following equations: saturation index = (a*2 + b*2)0.5; h° = arctg b* / a*. Color measurements were made at room temperature on the surface of each sample in triplicate at three randomly selected locations. 2.6.7. Texture measurements Texture profile analysis (TPA) was performed at room temperature with a Texture Analyzer TA-XT2i (Stable Micro Systems, Surrey, UK). Three cylindrical samples (2.5 cm 188 diameter) were taken from the middle of the semimembranosus muscle and subsequently were subjected to a two-cycle compression test. The samples were compressed to 40% of their original weight with a cylindrical probe of 5 cm diameter and a cross-head speed of 5 mm/s. Texture profile parameters were determined following descriptions by Bourne (1978) and the SMS manual (Stable Micro Systems, Surrey, UK). All analyses were performed in triplicate. 2.7. Statistical analysis The application of natural antioxidants (main variable under study) was performed individually to each ham (10 hams per treatment). Hence, each ham was considered an experimental unit and was successively subjected to a replicated process. Data obtained from the physicochemical and texture analyses of hams were evaluated by one-way Analysis of Variance (ANOVA). Tukey's test was performed when ANOVA revealed significant (P b 0.05) differences between formulations. The General Linear Model with repeated measures was used to analyze the data of meat color stability, lipid oxidation (TBARS) and protein oxidation (protein carbonyls concentration) over time of chilled storage. In the mixed statistical model, the individual ham was considered as a random effect, while the antioxidant treatment, the storage time (days 30 and 150) and the Treatment × Time interaction were considered as fixed effects. The Tukey's test was used for multiple comparisons of the means. The significance level was set at P b 0.05. SPSS (v. 18.0) software was used to carry out the statistic test. 3. Results and discussion 3.1. Proximate and fatty acid composition of cooked hams The proximate composition of cooked hams with added antioxidants at the end of the settling period is presented in Table 1. No significant differences among treatments were detected for the moisture, lipid, protein, sodium chloride, and heme iron. Cooked hams are characterized as a high protein product (19%) with low-fat content (3–5%) (Válková, Saláková, Buchtová, & Tremlová, 2007). The use of raw material from Iberian pigs in the manufacture of cooked hams resulted in an increase in the total amount of fat in the final product (N 5%). This higher fat content could affect the sensory quality of the final product (Jiménez-Colmenero, 2000). The relatively high heme iron content was consistent with those previously found in products manufactured with muscles from Iberian pigs (Estévez, Ventanas & Cava, 2007b; Utrera et al., 2012). While traditionally cooked meat products contain more than 2% of NaCl (Ruusunen & Puolanne, 2005), in our study the NaCl content went below that limit. Consistently, Ruusunen, Simolin, and Puolanne (2001) found similar results and observed that it is possible to reduce the salt content in cooked ham down to 1.7% NaCl without affecting its sensory properties. The knowledge of the pH values are of essential importance to predict the water retention capacity of cooked meat products, which has consequences on the yield and juiciness of the product (Aaslyng, Bejerholm, Ertbjerb, Bertram, & Andersen, 2003). The pH values were

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Table 1 Physico-chemical composition of cooked hams at the end of the settling period (30 days).

Moisture (g/100 g of sample) Sodium chloride (g/100 g of sample) Fat (g/100 g of sample) Protein (g/100 g of sample) pH awc Sodium nitrite (mg/100 g of sample) Heme iron (mg/100 g of sample)

Controla

Treatment 1b

Treatment 2b

Treatment 3b

68.72 ± 1.31 1.56 ± 0.39 4.50 ± 0.42 20.24 ± 1.41 5.99 ± 0.05a 0.97 ± 0.00a 0.07 ± 0.01a 0.83 ± 0.04b

69.05 ± 1.27 1.64 ± 0.33 5.38 ± 0.61 21.97 ± 0.96 5.87 ± 0.07a,b 0.95 ± 0.01c 0.07 ± 0.02a 0.94 ± 0.19a,b

70.65 ± 2.51 1.83 ± 0.35 5.32 ± 0.67 20.83 ± 1.07 5.80 ± 0.11b 0.96 ± 0.00b 0.05 ± 0.01b 1.06 ± 0.12a

68.64 ± 2.26 1.48 ± 0.26 5.10 ± 0.58 22.31 ± 1.20 5.77 ± 0.09c 0.97 ± 0.01a 0.06 ± 0.02a,b 1.10 ± 0.19a

Results are expressed as means ± standard error. Means with a different letter (a–c) within a same row are significantly different (P b 0.05). a Data partially published in Utrera et al. (2012). b Treatment 1: treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2: treated with Artinox® powder (3 g per kg of meat), and Treatment 3: treated with the experimental rose hip extract (300 mL per kg of meat). c aw: water activity.

within the range generally found in cooked meat products (5.77–5.87). The T3 showed significantly lower pH values than T1. This fact could be due to the composition of the extracts since phytochemicals from spices in T1 may have increased pH while the large concentration of ascorbic acid in T3 may have lowered pH in that group of hams. Similarly, the aw values were within the range generally reported for cooked meat products (0.95–0.97). The residual content of nitrite after 30 days of chilled storage was within the permissible values for this product (Directive 2006/52/EC of 5 July 2006). Cooked hams submitted to T2 had a significantly lower residual nitrite than T1 and T3. This fact could be due to the recognized ability of T2 components, namely erythorbate, to convert nitrite into nitrous oxide and hence, reduce the residual amount of the ingoing nitrite (Hasiak, Chaves, Sebranek, & Kraft, 1984). The fatty acid composition of cooked hams is shown in Table 2. The addition of three different sources of antioxidants had, as expected, no significant impact on the most abundant fatty acids at the end of the settling period. The amount of monounsaturated fatty acids (MUFA), saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) were

Table 2 Fatty acid profile of Iberian cooked hams at the end of the settling period (30 days).

C12:0 C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3 (n−3 + n−6) C20:0 C20:1 C20:2 C20:4 C20:5 (EPA) C22:6 (DHA) ΣSFAc ΣMUFAd ΣPUFAe

Controla

Treatment 1b

Treatment 2b

Treatment 3b

0.11 ± 0.02a 1.79 ± 0.15 24.08 ± 1.21 3.78 ± 0.78 0.39 ± 0.19 0.34 ± 0.11 10.79 ± 0.63 47.02 ± 2.28 9.18 ± 1.91 0.45 ± 0.09a

0.13 ± 0.02a 1.91 ± 0.15 24.24 ± 1.34 3.81 ± 0.38 0.26 ± 0.09 0.32 ± 0.13 10.59 ± 0.83 48.48 ± 2.47 8.28 ± 1.51 0.45 ± 0.08a

0.12 ± 0.04a 1.76 ± 0.21 24.00 ± 1.97 3.80 ± 0.46 0.51 ± 0.32 0.37 ± 0.07 10.80 ± 0.80 46.65 ± 2.33 9.19 ± 2.16 0.46 ± 0.09a

0.00 ± 0.00b 1.82 ± 0.15 23.86 ± 1.19 3.68 ± 0.88 0.32 ± 0.13 0.32 ± 0.10 10.81 ± 0.71 48.79 ± 2.80 7.81 ± 3.16 0.34 ± 0.15b

similar between treatments. As expected, oleic acid (C18:1) was the most abundant followed by palmitic (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6). Significant differences were found only between minority PUFA such as, linolenic acid, eicosatrienoic acid and docosahexenoic acid (DHA). These differences may be derived from presence of such fatty acids in the extracts from the diverse vegetable sources. The fatty acid profile of meat products is relevant as it influences many quality parameters among which are the oxidative stability, sensory quality and also the nutritional value of the final meat product (Wood et al., 2004). 3.2. Lipid oxidation Fig. 1 shows the evolution of TBARS values through chilled storage (30–150 days). Compared to the values from the Control cooked hams, T2 and T3 had a significant effect on the extent of lipid oxidation after 30 days settling. In all samples, however, the TBARS values remained considerably low (b0.2 mg MDA/kg sample) and far from values that may reflect rancidity perception (Greene & Cumuze, 1982). TBARS values increased from 30 to 150 days in Control, T2 and T3. After the entire settling period (150 days) TBARS numbers were significantly lower in T1 samples than in the T2 and T3 counterparts. According to these results, the significant increase of TBARS observed in T2 and T3 during storage was efficiently inhibited by the mixture of spices added to T1. The mixture of rosemary, clove, cinnamon and garlic essential oils (Treatment 1) has more powerful antioxidant effects against lipid oxidation than the other antioxidants used in this study. The abundance of compounds with antioxidant activity in these spices, especially

0.11 ± 0.03 0.11 ± 0.02 0.10 ± 0.06 0.12 ± 0.02 0.71 ± 0.06 0.72 ± 0.08 0.70 ± 0.07 0.70 ± 0.06 0.29 ± 0.17b 0.36 ± 0.27a 0.21 ± 0.03c 0.21 ± 0.03c 0.43 ± 0.48b 1.25 ± 0.69a 1.10 ± 0.46a 0.51 ± 0.49b 0.05 ± 0.02 0.07 ± 0.03 0.08 ± 0.05 0.06 ± 0.04 0.06 ± 0.05b 0.05 ± 0.02a 0.14 ± 0.09a 0.09 ± 0.06a,b 36.89 ± 2.98 37.25 ± 2.45 37.28 ± 3.40 36.93 ± 2.20 51.85 ± 2.85 53.33 ± 3.05 51.53 ± 2.93 53.49 ± 3.84 10.53 ± 3.07 9.73 ± 2.46 11.27 ± 3.07 9.57 ± 3.89

Results are expressed as means ± standard error. (a–c) Means with different letters superscript in the same row are significantly different (P b 0.05). a Data partially published in Utrera et al. (2012). b Treatment 1: treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2: treated with Artinox® powder (3 g per kg of meat), and Treatment 3: treated with the experimental rose hip extract (300 mL per kg of meat). c Saturated fatty acids. d Monounsaturated fatty acids. e Polyunsaturated fatty acids.

Fig. 1. Evolution of TBARS numbers in cooked hams at 30–150 days of chilled storageA. Different letters on top of bars denote significant differences between treatments-times combinations (P b 0.05). AData from Control group partially published in Utrera et al. (2012). Treatment 1: treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2: treated with Artinox® powder (3 g per kg of meat), and Treatment 3: treated with the experimental rose hip extract (300 mL per kg of meat).

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those obtained from rosemary and cloves such as carnosol or rosmarinic acid, could be responsible for their antioxidant activity (Mancini-Filho, Van-Koiij, Mancini, Cozzolino, & Torres, 1998; Yu, Scanlin, Wilson, & Schmidt, 2002). Previous studies have demonstrated that phytochemicals from these spices have radical scavenging and metal chelating properties which could explain the stabilizing effect on lipids (Dwivedi et al., 2006; Nissen et al., 2004). In fact, Kong, Zhang, and Xiong (2010) reported that the ability of cloves and rosemary to chelate metals is enhanced at higher concentrations of the spices. Additionally, several other studies have shown that these spices have the same effectiveness against lipid oxidation in cooked meat products than synthetic antioxidants such as BHT (Estévez et al., 2005; Jawahar, Balasundari, Indra, & Jeyachandran, 1994). However, taking into consideration the considerably low lipid oxidation rates found in the present samples, the addition of curing agents (nitrite/ascorbate) in the basic brine formulation may have been enough to control this reaction and its negative consequences. Furthermore, the exclusion of oxygen in the vacuum packing applied to products during storage (10 mbar pressure) may also have contributed to keeping low TBARS values in the present samples. 3.3. Protein oxidation Fig. 2 shows the results from the analysis of the oxidative deterioration of proteins during chilled storage (30–150 days). After the first settling period (30 days) the amount of carbonyls in the three samples under study (ranging from 1.72 to 3.21 nmol carbonyls/mg protein) was significantly lower than that of the Control counterparts (7.88 nmol carbonyls/mg protein). Among the antioxidant treatments, T2 and T3 appeared as the most effective against protein oxidation. The antioxidant activity of plant phenolics against protein oxidation has previously been described in model systems and cooked meat products like frankfurters and liver pâté (Estévez & Heinonen, 2010; Estévez, Ramírez, et al., 2007; Estévez, Ventanas, et al., 2007). In particular, rose hips have been reported to be particularly efficient against protein carbonylation in processed porcine patties and frankfurters due to the high concentration of ascorbic acid and other redox-active phenolic compounds (Armenteros, Morcuende, Ventanas, & Estévez, 2013; Ganhão, Estévez, Armenteros & Morcuende, 2013; Ganhão, Estévez, Kylli, Heinonen & Morcuende, 2010; Ganhão, Morcuende, et al., 2010; Vossen, Utrera, De Smet, Morcuende, & Estévez, 2012). A recent study proved the remarkable ability of ascorbate to protect myofibrillar proteins against hydroxyl radicals (Villaverde, Parra, & Estévez, 2014). On the other hand, nitrite, present in all samples, is known to be a highly efficient inhibitor of lipid oxidation (Honikel, 2008) but had a negligible

Fig. 2. Total carbonyl content in cooked hams at 30–150 days of chilled storageA. Different letters on top of bars denote significant differences between treatments-times combinations (P b 0.05). AData from Control group partially published in Utrera et al. (2012). Treatment 1: treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2: treated with Artinox® powder (3 g per kg of meat), and Treatment 3: treated with the experimental rose hip extract (300 mL per kg of meat).

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effect against meat protein oxidation (Villaverde et al., 2014). The present study corroborates that a natural source of ascorbate (rose hips) is able to protect proteins against carbonylation in an intact cooked meat product. Thus, it is reasonable that erythorbate (ascorbate isomer) also had a similar antioxidant effect against the formation of protein carbonyls in T2 samples. Nitrite and ascorbate from the basic brine formulation, present in all samples, may have protected lipids against oxidation, explaining the low TBARS found in the present samples. The lack of effect of these species at the basal levels on myofibrillar proteins leaves some space for other substances to show further antioxidant effect on meat proteins. These results reflect the complex and different chemistry behind lipid and protein oxidation as profusely reported in recent reviews (Bekhit, Hopkins, Fahri, & Ponnampalam, 2013; Estévez, 2011; Soladoye, Juarez, Aalhus, Shand, & Estévez, 2015). On the other hand, phenolic diterpenes, certain phenolic acids, flavonols, and tea catechins have also been previously found to inhibit protein oxidation in meat products as measured by the DNPH method (reviewed by Falowo, Fayemi, & Muchenje, 2014). These phenolic compounds could have acted as iron chelators and scavengers of lipid-mediated ROS which very likely are main initiators of protein carbonylation in meat systems (Estévez, 2011). While a consecutive increase of protein carbonyls was expected during the subsequent storage (from day 30 to day 150), a reduction of protein carbonyls was observed instead in T1 samples. The loss of protein carbonyls in processed and stored muscle foods has been described before and may respond to the involvement of these reactive species in further reactions, including formation of carboxylic acids and Schiff base structures (Estévez, 2011; Vossen et al., 2012). The fact that protein carbonyls are not accumulated as stable end oxidation products limits the suitability of using these compounds as reliable markers of protein oxidation in processed meat products (Soladoye et al., 2015). 3.4. Instrumental color of cooked hams Table 3 shows the evolution of the color parameters of cooked hams after a settling period (30 days) and at the end of the chilled storage (150 days). After the settling period (30 days), cooked hams treated with the sources of antioxidants displayed significantly lower values of redness (a*) and significantly higher of lightness (L*) and yellowness (b*) than Control hams. Among treatments, T2 had in turn, significantly

Table 3 Instrumental color of cooked hams at 30–150 days. Controla

Treatment 1a

Treatment 2a

Treatment 3a

L* Day 30 Day 150

65.98 ± 4.25a 72.98 ± 7.25a

42.30 ± 8.79c 53.48 ± 14.66b

28.38 ± 4.83d 42.98 ± 11.77c

39.37 ± 6.20 c 40.51 ± 14.41c

a* Day 30 Day 150

10.10 ± 1.62b 9.23 ± 1.02b

13.15 ± 2.51a 14.39 ± 2.22a

10.74 ± 1.96b 13.91 ± 2.70a

13.34 ± 2.67a 13.60 ± 3.50a

b* Day 30 Day 150

7.25 ± 0.72a 8.39 ± 1.23a

5.09 ± 1.13b 5.30 ± 1.80b

3.57 ± 0.70 c 3.96 ± 1.12c

Chroma Day 30 Day 150

15.82 ± 1.29a 16.01 ± 2.18a

14.12 ± 2.67a 15.38 ± 2.64a

11.35 ± 1.87b 14.49 ± 2.79a

14.16 ± 2.86a 14.37 ± 3.81a

Hue Day 30 Day 150

27.18 ± 3.13a 29.98 ± 2.83a

21.14 ± 2.89b 19.77 ± 4.08bc

18.88 ± 4.68 bc 15.91 ± 3.62c

19.27 ± 3.12 bc 18.51 ± 2.65bc

Color parameters

4.69 ± 1.28bc 4.62 ± 1.63bc

Results are expressed as means ± standard error. (a–c) Means with different letters indicate significant differences (P b 0.05) between all 8 time-treatments combinations. A Data partially published in Utrera et al. (2012). a Treatment 1: treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2: treated with Artinox® powder (3 g per kg of meat), and Treatment 3: treated with the experimental rose hip extract (300 mL per kg of meat).

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Table 4 Instrumental texture of cooked hams at the end of the settling period (30 days).

Hardness (N) Adhesiveness (Nxs) Springiness (cm) Cohesiveness (dimensionless) Gumminess (N) Chewiness (N) Resilience (dimensionless)

ControlA

Treatment 1B

Treatment 2B

Treatment 3B

28.43 ± 3.72b −28.82 ± 20.90 0.82 ± 0.09 0.50 ± 0.06 14.25 ± 2.04b 12.15 ± 2.60 b 0.32 ± 0.09

67.36 ± 21.25a −30.78 ± 21.10 0.81 ± 0.12 0.53 ± 0.07 35.56 ± 12.24a 29.41 ± 11.32 a 0.31 ± 0.09

62.93 ± 17.94a −27.32 ± 20.97 0.85 ± 0.10 0.53 ± 0.07 33.85 ± 12.15a 29.08 ± 11.61a 0.31 ± 0.08

59.41 ± 16.63a −34.42 ± 25.04 0.82 ± 0.09 0.54 ± 0.05 32.32 ± 10.65a 26.85 ± 10.58a 0.30 ± 0.07

Results are expressed as means ± standard error. (a–b) Means with different letters within the same row were significantly different (P b 0.05). A Data partially published in Utrera et al. (2012). B Treatment 1: treated with essential oils of garlic, cinnamon, cloves and rosemary (1 g per kg of meat); Treatment 2: treated with Artinox® powder (3 g per kg of meat), and Treatment 3: treated with the experimental rose hip extract (300 mL per kg of meat).

lower values of the aforementioned color parameters than the other two treatments. The lightness (L *) is considered to govern the quality of meat products and the best predictor of the pink color while hue angle provides information about the real true red color in muscle foods (Brewer, Zhu, Bidner, Meisinger, & McKeith, 2001). According to the present results, the color reaction was clearly improved by the presence of the antioxidants, in particular by the T2 treatment. The subsequent refrigerated storage led to a significant modification of the color parameters leading to general increases of lightness and decreases of the hue angle. Unlike the other two treatments, samples submitted to T3 suffer no significant color changes. In the present study, the addition of the rose hips extracts led to an efficient inhibition of the color deterioration, likely caused by the control of oxidative reactions. The protective effect of this natural extract against discoloration can be ascribed to the high concentration of phenolic compounds which are naturally present in these fruits (Armenteros et al., 2013; Ganhão, Estévez, et al., 2010; Vossen et al., 2012). The addition of substances with antioxidant activity could inhibit the discoloration of cooked ham during chilled storage as it has been described for other similar cooked muscle foods (Armenteros et al., 2013; Ganhão, Morcuende, et al., 2010; 2013; Keokamnerd, Acton, Han, & Dawson, 2008). This effect has been previously observed for other spices in fresh pork patties (Jo, Son, Son, & Byon, 2003) and cooked beef burgers (Tang, Kerry, Sheehan, Buckley, & Morrissey, 2001). 3.5. Texture profile analysis of cooked hams The values of texture parameters at the end of the settling period (30 days) are shown in Table 4. No significant differences were found between treatments. However, the overall texture values in these samples contrast with the data reported by Utrera et al. (2012) in cooked hams with no added antioxidants. In that previous study, the hardness, gumminess and chewiness of the samples were considerably lower than the values reported in the present study. Similar results were reported by Estévez et al. (2005) and Utrera, Morcuende, Ganhão, and Estévez (2015) in frankfurters and cooked patties treated with rosemary essential oil and rose hips extracts, respectively. These authors linked the increase of hardness in the treated products to the prooxidant effect of the phytochemicals on meat proteins. In particular, the increased carbonylation led to the formation of cross-links via Schiff base formation which was thought to be responsible for the strengthening of the meat structure. This pro-oxidant effect was not observed in the present study and Schiff bases, in particular, were not measured in the present samples. Besides this likely mechanism, the precise means by which the enhancement of cooked hams with brine enriched in phytochemicals leads to increases of hardness remains indefinite. 4. Conclusions The application of natural sources of antioxidants to cooked hams did not lead to significant changes in the physico-chemical parameters analyzed. While lipid oxidation remained at low rates, protein oxidation

levels were considerably high after settling and particularly after the subsequent chilled storage. To this regard, R. canina L. extracts (T3) were the most effective in controlling protein carbonylation at the end of storage (150 days) as a likely result of the presence of ascorbate. The application of these antioxidants via brine injection may be a feasible way of inhibiting protein oxidation and their negative consequences in cooked hams. The potential negative influence of these treatments on texture parameters should be addressed in further studies. Acknowledgments This work was supported by the CDTI project (IDI 20090264). We thank the owners, managers and staff of Consorcio de Jabugo, S.A. Mario Estévez thanks the Spanish Ministry of Economy and Competitiveness for the contract RYC-2009-03901 and the project AGL2010-15134. The scientific advice of Antonio Silva (SiPA, University of Extremadura) is also acknowledged. References Aaslyng, M. D., Bejerholm, C., Ertbjerb, P., Bertram, H., & Andersen, H. J. (2003). Cooking loss and juiciness of pork in relation to raw meat quality and cooking procedure. Food Quality and Preference, 14(4), 277–288. Aguirrezábal, M. M., Mateo, J., Domínguez, M. C., & Zumalacárregui, J. M. (2000). Garlic was as effective as the mixture of additives in inhibiting lipid oxidation. Meat Science, 54, 77–81. Ahn, J., Grün, I. U., & Mustapha, A. (2007). Effects of plant extracts on microbial growth, color change and lipid oxidation in cooked beef. Food Microbiology, 24(1), 7–14. AOAC (2000). Official methods of analysis (17th ed.). Gaithersburgh. Maryland: Association of Official Analytical Chemists. Armenteros, M., Morcuende, D., Ventanas, S., & Estévez, M. (2013). Application of natural antioxidants from strawberry tree (Arbutus unedo L.) and dog rose (Rosa canina L.) to frankfurters subjected to refrigerated storage. Journal of Integrative Agriculture, 12, 1972–1981. Bekhit, A. E. -D. A., Hopkins, D. L., Fahri, F. T., & Ponnampalam, E. N. (2013). Oxidative processes in muscle systems and fresh meat: Sources, markers, and remedies. Comprehensive Reviews in Food Science and Food Safety, 12, 565–597. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Bourne, M. C. (1978). Texture profile analysis. Food Technology, 33, 62–66. Brewer, M. S., Zhu, L. G., Bidner, B., Meisinger, D. J., & McKeith, F. K. (2001). Measuring pork color: Effects of bloom time, muscle, pH and relationship to instrumental parameters. Meat Science, 57, 169–176. Cando, D., Morcuende, D., Utrera, M., & Estévez, M. (2014). Phenolic-rich extracts from willowherb (Epilobium hirsutum L.) inhibit lipid oxidation but accelerate protein carbonylation and discoloration of beef patties. European Food Research and Technology, 238, 741–751. Craig, J. W. (1999). Health-promoting properties of common herbs. The American Journal of Clinical Nutrition, 70, 491S–499S. Delahunty, C. M., McCord, A., O'Neill, E. E., & Morrissey, P. A. (1997). Sensory characterisation of cooked ham by untrained consumers using free-choice profiling. Food Quality and Preference, 8, 381–388. Djenane, D., Sánchez-Escalante, A., Beltrán, J. A., & Roncalés, P. (2003). Extension of the shelf life of beef stakes packaged in a modified atmosphere by treatment with rosemary and displayed under UV-free lighting. Meat Science, 64, 417–426. Dwivedi, S., Vasavada, M., & Cornforth, D. (2006). Sensory attributes of Chinese 5-spice ingredients in cooked ground beef. Journal of Food Science, 71(1), C12–C17. Estévez, M. (2011). Protein carbonyls in meat systems: A review. Meat Science, 89, 259–279. Estévez, M., & Heinonen, M. (2010). Effect of phenolic compounds on the formation of αamoniadipic and γ-glutamic semialdehydes from myofibrillar proteins oxidized by

M. Armenteros et al. / Meat Science 116 (2016) 253–259 cooper, iron, and myoglobin. Journal of Agricultural and Food Chemistry, 58, 4448–4455. Estévez, M., Ramírez, R., Ventanas, S., & Cava, R. (2007a). Sage and rosemary essential oils versus BHT for the inhibition of lipid oxidative reactions in liver pate. LWT- Food Science and Technology, 40, 58–65. Estévez, M., Ventanas, S., & Cava, R. (2007b). Oxidation of lipids and proteins in frankfurters with different fatty acid compositions and tocopherol and phenolic contents. Food Chemistry, 100, 55–63. Estévez, M., Morcuende, D., & Cava, R. (2005). Protein oxidation in frankfurters with increasing levels of added rosemary essential oil: Effect on color and texture deterioration. Journal of Food Science, 70, 427–432. Falowo, A. B., Fayemi, P. O., & Muchenje, V. (2014). Natural antioxidants against lipid– protein oxidative deterioration in meat and meat products: A review. Food Research International, 64, 171–181. Ganhão, R., Morcuende, D., & Estévez, M. (2010a). Protein oxidation in cooked burger patties with added fruit extracts: Influence on colour and texture deterioration during chill storage. Meat Science, 85, 402–409. Ganhão, R., Estévez, M., Kylli, P., Heinonen, M., & Morcuende, D. (2010b). Characterization of selected wild Mediterranean fruits and comparative efficacy as inhibitors of oxidative reactions in emulsified raw pork burger patties. Journal of Agricultural and Food Chemistry, 58, 8854–8861. Ganhão, R., Estévez, M., & Morcuende, D. (2011). Suitability of the TBA method for assessing lipid oxidation in a meat system with added phenolic-rich materials. Food Chemistry, 126, 772–778. Ganhão, R., Estévez, M., Armenteros, M., & Morcuende, D. (2013). Mediterranean berries as inhibitors of lipid oxidation in porcine burger patties subjected to cooking and chilled storage. Journal of Integrative Agriculture, 12, 1982–1992. Greene, B. E., & Cumuze, T. H. (1982). Relationship between TBA numbers and inexperienced panelists'assessments of oxidized flavor in cooked beef. Journal of Food Science, 47, 52–54. Hasiak, R. J., Chaves, J., Sebranek, J., & Kraft, A. A. (1984). Effect of sodium nitrite and sodium erythorbate on the chemical, sensory, and microbiological properties of water-added turkey ham. Poultry Science, 63, 1364–1371. Honikel, K. O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Science, 78, 68–76. Ito, M., Murakami, K., & Yoshino, M. (2005). Antioxidant action of eugenol compounds: Role of metal ion in the inhibition of lipid oxidation. Food and Chemical Toxicology, 43, 461–466. Jawahar, A. T., Balasundari, S., Indra, J. G., & Jeyachandran, P. (1994). Influence of antioxidants on the sensory quality and oxidative rancidity. Journal of Food Science and Technology India, 31, 168–170. Jayathilakan, K., Sharma, G. K., Radhakrishna, K., & Bawa, A. S. (2007). Antioxidant potential of synthetic and natural antioxidants and its effect on warmed-over-flavour in different species of meat. Food Chemistry, 105, 908–916. Jiménez-Colmenero, F. (2000). Relevant factors in strategies for fat reduction in meat products. Trends in Food Science & Technology, 11(2), 56–66. Jo, C., Son, J. H., Son, C. B., & Byon, M. W. (2003). Functional properties of raw and cooked pork patties with added irradiated freeze-dried green tea leaf extract powder during storage at 4 °C. Meat Science, 64, 13–17. Kanner, J. (1994). Oxidative process in meat and meat products: Quality implications. Meat Science, 36, 169–189. Keokamnerd, T., Acton, J. C., Han, I. Y., & Dawson, P. L. (2008). Effect of commercial rosemary oleoresin preparations on ground chicken though meat quality, packaged in a high-oxygen atmosphere. Poultry Science Association, 87, 170–179. Kong, B., Zhang, H., & Xiong, X. L. (2010). Antioxidant activity of spice extracts in a liposome system and in cooked pork patties and the possible mode of action. Meat Science, 85, 772–778. López-Bote, C. J., Rey, A., Sanz, M., Gray, J. L., & Buckley, J. D. (1997). Dietary vegetable oils and α-tocopherol reduce lipid oxidation in rabbit muscle. Journal of Nutrition, 127, 1176–1182.

259

Mancini-Filho, J., Van-Koiij, A., Mancini, D. A. P., Cozzolino, F. F., & Torres, R. P. (1998). Antioxidant activity of cinnamon (Cinnamomun zeylanicum, Breyne) extracts. Bollettino Chimico Farmaceutico, 137, 443–447. McCarthy, T. L., Kerry, J. P., Kerry, J. F., Lynch, P. B., & Buckley, D. J. (2001). Evaluation of the antioxidant potential of natural food/plant extracts as compared with synthetic antioxidants and vitamin E in raw and cooked pork patties. Meat Science, 58, 45–52. Murcia, A. M., Egea, I., Romojaro, F., Parras, P., Jimenez, A. M., & Martinez-Tome, M. (2004). Antioxidant evaluation in dessert spices compared with common food additives. Influence of irradiation procedure. Journal of Agricultural and Food Chemistry, 52(7), 1872–1881. Nissen, L. R., Byrne, D. V., Bertelsen, G., & Skibsted, L. H. (2004). The antioxidative activity of plant extracts in cooked pork patties as evaluated by descriptive sensory profiling and chemical analysis. Meat Science, 68, 485–495. Oliver, C. N., Ahn, B. W., Moerman, E. J., Goldstein, S., & Stadtman, E. R. (1987). Agedrelated changes in oxidized proteins. Journal of Biological Chemistry, 262, 5488–5491. Rodríguez-Carpena, J. G., Morcuende, D., & Estévez, M. (2012). Avocado, sunflower and olive oils as replacers of pork back-fat in burger patties: Effect on lipid composition. Oxidative stability and quality traits. Meat Science, 90, 106–115. Ruusunen, M., & Puolanne, E. (2005). Reducing sodium intake from meat products. Meat Science, 70, 531–541. Ruusunen, M., Simolin, M., & Puolanne, E. (2001). The effect of fat content and flavor enhancers on the perceived saltiness of cooked “Bologna-type” sausages. Journal of Muscle Foods, 12, 107–120. Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Soladoye, O. P., Juarez, M. L., Aalhus, J. L., Shand, P., & Estévez, M. (2015). Protein oxidation in processed meat: Mechanisms and potential implications on human health. Comprehensive Reviews in Food Science and Food Safety, 14, 106–122. St. Angelo, A. J., Vercelloti, J. R., Vinnett, C. H., Kuan, J. W., James, C., Jr., & Dupuy, H. P. (1987). Chemical and instrumental analyses of warmed-over flavor in beef. Journal of Food Science, 52, 1163–1168. Tang, S., Kerry, J. P., Sheehan, D., Buckley, D. J., & Morrissey, P. A. (2001). Antioxidative effect of added tea catechins on susceptibility of cooked red meat, poultry and fish patties to lipid oxidation. Food Research International, 34, 651–657. Toldrá, F., Mora, L., & Flores, M. (2010). Cooked ham. Handbook of meat processing (pp. 301–312). Oxford, Uk: Willey-Blackwell. Utrera, M., Armenteros, M., Ventanas, S., Solano, F., & Estévez, M. (2012). Pre-freezing raw hams affects quality traits in cooked hams: Potential influence of protein oxidation. Meat Science, 92, 596–603. Utrera, M., Morcuende, D., Ganhão, R., & Estévez, M. (2015). Role of phenolics extracting from Rosa canina L. on meat protein oxidation during frozen storage and beef patties processing. Food and Bioprocess Technology, 8, 854–864. Válková, V., Saláková, A., Buchtová, H., & Tremlová, B. (2007). Chemical, instrumental and sensory characteristics of cooked pork ham. Meat Science, 77, 608–615. Villaverde, A., Parra, V., & Estévez, M. (2014). Oxidative and nitrosative stress induced in myofibrillar proteins by a hydroxyl-radical-generating system: Impact of nitrite and ascorbate. Journal of Agricultural and Food Chemistry, 62, 2158–2164. Vossen, E., Utrera, M., De Smet, S., Morcuende, D., & Estévez, M. (2012). Dog rose (Rosa canina L.) as a functional ingredient in porcine frankfurters without added sodium ascorbate and sodium nitrite. Meat Science, 92, 451–457. Wood, J. D., Richardson, R. I., Nute, G. R., Fisher, A. V., Campo, M. M., Kasapidou, E., ... Enser, M. (2004). Effects of fatty acids on meat quality: A review. Meat Science, 66, 21–32. Yang, G. C., Yasaei, P. M., & Page, S. W. (1993). Garlic as anti-oxidants and free radical scavengers. Journal of Food and Drug Analysis, 14, 357–364. Yoo, K. M., Lee, C. H., Lee, H., Moon, B., & Lee, C. Y. (2008). Relative antioxidant and cytoprotective activities of common herbs. Food Chemistry, 106, 929–936. Yu, L., Scanlin, L., Wilson, J., & Schmidt, G. (2002). Rosemary extracts as inhibitors of lipid oxidation and color change in cooked turkey products during refrigerated storage. Journal of Food Science, 67, 582–585.