Effect of Origanum syriacum L. Essential Oil on the Storage Stability of Cooked Chicken Meat

28/December/2017 Approved: 16/July/2018 ABSTRACT The effects of the addition of Origanum syriacum L. essential oil (OE) on the lipid and protein oxidation, and sensory attributes of cooked chicken meat were compared with those of synthetic commercial meat preservatives. Ground deboned and skinless chicken breast and thigh meat were distributed according to six treatments: (T1) control (no addition of meat preservative); or the addition of (T2) 100 ppm OE; (T3) 150 ppm OE; (T4) 300 ppm L-ascorbic acid (E-300); (T5) 5 and 14 ppm butylated hydroxyanisoleadded to breast and thigh meat, respectively, (BHA/E-320); and (T6) 150 ppm sodium nitrite (E-250). Meat samples were cooked and analyzed for lipid oxidation (TBARS levels) and protein oxidation (carbonyl levels) on days 0, 4, and 7 days of storage. In addition, cooked meat thigh patties were evaluated for cooking loss and sensory attributes. All additives were showed significant lipid and protein antioxidant effects ( p< 0.05) compared with the control treatment during storage, with the strongest effects obtained with OEat 150 ppm and E-250. Cooking loss was not influenced ( p> 0.05) by the treatments. The best sensory attribute scores were obtained with OEat 150 ppm and E-250 treatments. L-ascorbic acid and BHA also showed significant effect ( p< 0.05) on both lipid and protein oxidation values, and sensory attributes. Based on the results study, it concluded that OEat 150 ppm may be used in replacement of synthetic antioxidants to improve the storage stability


INTRODUCTION
Synthetic antioxidants, such as butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT) and tertiary butyl-hydroquinone (TBHQ) were widely used in several countries such as United States of America (USA) as an effective antioxidant in poultry meat products (US-FDA, 2016a). On the other hand, in the Middle East, countries such as Jordan, which follow the European Commission regulations (Codex Alimentarius) have limitations on the use of synthetic antioxidants (SA) in their food (JSMO, 2016). However, all over the world, there is a negative impression on the use of SA due to their possible toxicological and carcinogenic effects (Kumar et al., 2015;Altmann et al., 1986). Therefore, natural antioxidants (NA) have been increasingly replaced SA by the meat processing industry, and are preferred due to recent changes in consumers' demand (Taghvaei & Jafari, 2015;Abdel-Hamied et al., 2009).
In addition to SA, sodium nitrite (E-250) has been used as meat preservative and curing ingredient to improve meat storage stability (Al-Shuibi & Al-Abdullah, 2002). Meat processing companies extensively usedE-250 as a curing agent, as labeled in their food products. E-250 is also a very important meat additive as it improves cooked-meat color, flavor, and storage stability (Honikel, 2008;Sindelar & Milkowski,

Effect of Origanum syriacum L. Essential Oil on the Storage Stability of Cooked Chicken Meat
eRBCA-2019-0719 2011). However, it may negatively affect human health when present in meat due to the formation of nitrosamines. Previous research and epidemiological studies (Sebranek & Bacus, 2007;Oostindjer et al., 2014) indicated that these N-Nitroso compounds maybe carcinogenic, causing colorectal cancer in humans. Meat processers have changed their curing formulations to reduce the use of E-250 by increasing sodium nitrate levels and mixing it with ascorbate or erythorbate to maximize curing reaction (Oostindjer et al., 2014;Nunez De Gonzalez et al., 2012). The addition of these accelerators (reducers) also may inhibit the formation of nitrosamines (Tannenbaum & Leaf, 1991). Therefore, in order to increase the antioxidant capacity of processed meat, E-250is combined with E-300or other SA (Al-Shuibi & Al-Abdullah, 2002). Furthermore, E-300 is permitted by the European Commission to be used as an antioxidant in different meat products to extend their shelf life and improve their stability (Velasco & Williams, 2011).
According to the recent publications (Kumar et al., 2015;Abdel-Hamied et al., 2009), a novel approach is to find NA for the complete or partial substitution of SA. For instance, the direct inclusion of herbs and their essential oil extracts, including sage, rosemary, and green tea, in meat products have shown positive overall effects on meat quality and their storage stability (Abdel-Hamied et al., 2009). Oregano essential oil (OE) is one of the most promising (ranks the highest) herbal extracts evaluated to improve meat storage stability (Zheng & Wang, 2001). The essential oil of Origanum syriacum L. (commonly known as Za'atar) is characterized by its high thymol and carvacrol contents (Daouk et al., 1995;Tepe et al., 2004;Ibrahim et al., 2012a).In Jordan and the Middle Eastin general, Origanum syriacum L. ground leaves are dried and consumed in many different traditional ways. In addition, Origanum syriacum leaves are used to cure eye ailments, burns, and stomach conditions, whereas its seeds are used as a sedative (Oran & Al-Eisawi, 1998;Ibrahim et al., 2012a).
Origanum syriacum essential oil provides a pleasant flavor to meat, reduces off-odor, and prevents rancidity development. These properties called the attention of researchers on meat preservation technology. Studies showed that Origanum syriacum L. has very strong antioxidant effect due to the high levels of thymol and carvacrol in their composition (Adam et al., 1998;Yanishlieva et al., 1999;Tepe et al., 2004;Al-Bandak, 2007) compared with the other Origanum species, such as Origanum vulgare, Origanumonties, and Origanummajorana L. (Daouk et al., 1995;Berna'th, 1997;Baser, 2002;Sahin et al., 2004). The antioxidant activity of OE is affected by several factors, including genetics, season, temperature, moisture, soil, and daylight length (Ibrahim et al., 2012b;Farooqi et al., 1999;Gonuz & Ozorgucu, 1999;Ceylan et al., 2003;Viuda-Martos et al., 2010). The genetic variation among Origanum species is considered most important factor of variation of essential oil percentage and composition (Akeel et al., 2009). However, the food preservation properties of wild Jordanian OE need to be further investigated. In addition, studies comparing essential oils extracted from different Origanum species, which are characterized by different phenolic compound contents, are needed. For instance, Origanum syriacum var. bevanii collected in Turkey contains 43-79% carvacrol (Baser, 2002). Also, the inclusion levels OE as an alternative to the most popular meat preservatives still need evaluated.
The hypothesis of this study was that wild Origanum syriacum L. found In Jordan may have strong antioxidant effect, allowing it to be applied as a natural alternative to synthetic antioxidants.
The objectives of this study were: 1) to investigate of two different Origanum syriacum L. essential oil levels on the storage stability and quality characteristics of ground and cooked during storage, and 2) to compare the antioxidant effect of Origanum syriacum L. with the most common antioxidants used in the meat industry.

Sample preparation
The experimental procedures were reviewed and approved by the Department of Animal Production, Agriculture College, Mu'tah University, Jordan. The health and welfare of the experimental birds were checked by a veterinarian.
One hundred and forty, 6-wk-old broilers fed a corn-soybean meal diet were slaughtered at the experimental facilities of Mu'tah University, according to using the standard guidelines for poultry slaughter of Jordan (Jordanian Ministry of Agriculture Guidelines). Carcasses were chilled in iced water for 2 h, and drained in a cold chamber. The muscles (breast and thigh) were separated from the carcasses 24h after slaughter. Boneless breast and thigh muscles were cleaned, skins removed, external fats trimmed off, vacuum packed in oxygen-impermeable bags, and stored at -18 o C freezer until used.
The frozen meats were thawed in a cooler (4°C), and ground twice using a 10-mm and a 3-mm plates (Moulinex, Type DKA1, France) before use. Meat samples were submitted to the following six treatments:1) no additive inclusion, 2) addition of 100 ppm OE (OE-100), 3) addition of 150 ppm OE (OE-150), 4) addition of 300 ppm E-300, 5) addition of 5 and 14ppm BHA to the breast and thigh meat, respectively, and 6) addition of 150 ppm E-250.
Oregano essential oil (pure extract) was obtained from a certified company in Jordan (Green Fields Factory for oils, Amman, Jordan) using the most efficient purification, extraction, and steam distillation methods. This essential oil was extracted from the wild Origanum syriacum L. collected from different regions in Jordan. The HPLC analysis of the OE (Royal Scientific Society, Jordan, Amman), using the method of Zekovic et al. (2000), indicated that the essential oil consisted of 76.39% of carvacrol, and 13.06%of thymol. L-ascorbic acid (Fisher Scientific, fair Lawn, N.J., USA), and E-250 (Gainland Chemical Company, UK) powder were first dissolved in de-ionized distilled water (DDW), then the oil emulsion a (water in oil), using mineral oil, were prepared to make their aqueous solution. BHA and OE were dissolved in 100% ethanol, and then mixed with mineral oil to make their stock solutions. The added ethanol was removed in a rotary evaporator (model Laborota 4001-efficient, Heidolph, Germany) at 70°C, 175 mbar vacuum pressure, before adding the stock solution to meat samples. Each additive was added to the ground breast or thigh meat and then mixed for 3 min in a bowl mixer. All additives contained the same amounts of mineral oil and water (oil emulsion) to provide the same conditions.
The raw meat samples were packed in oxygenimpermeable vacuum bags (Albalabki-Jordan, Malcom SRL, Italy), and were cooked in-bag in 90°C water bath (WNB 14, Memmert, Germany) until the internal temperature of the meat patties reached 75°C. After cooling to room temperature, the cooked meat samples (approximately 50g) were transferred to oxygenpermeable bag (polyethylene, 11 x 25 cm, Future for Plastic Industry, Al-Moumtaz bags, Co. L.T.D, Amman, Jordan), and stored at 4°C for up to 7 days, when lipid and protein oxidation, cooking loss, and sensory analyses were carried out. The ground chicken (raw thigh)meat patties stored at 4°C up to 4 days before cooking and for each evaluation session.

Thiobarbituric acid-reactive substances (TBARS) measurement
Lipid oxidation of the meat samples was determined using the TBARS method (Ahn et al., 1998) with minor modifications. Five grams of ground chicken meat were weighed, placed in a 50-mL test tube, 50 μL BHT (7.2%) and 15 mL deionized distilled water (DDW) were added, and homogenized in a Heidloph (DIAX-900, Germany) for 25s at high speed. One milliliter of the meat homogenate was then transferred to a disposable test tube (13 × 100 mm), to which thiobarbituric acid/trichloroacetic acid solution (15 mM TBA/15% TCA, 2 mL) was added. The mixture was vortexed and incubated in water bath (90°C) for 15 min when the pink color was developed. Test tubes were then cooled in iced water for 10 min, mixed again, and centrifuged for 15 min at 2,500 x g at 4°C. The absorbance of the resulting supernatant solution was confirmed at 532 nm against a blank containing 1 mL of DDW and 2 mL of TBA/TCA solution. The TBARS number was expressed as mg of malondialdehyde (MDA) per kg of meat.

Protein oxidation
Protein oxidation was determined by the general method of total carbonyl value proposed by Lund et al. (2008). In a test tube, 10 mL pyrophosphate buffer (2.0 mM Na 4 P 2 O 7 , 10 mM Trizma-maleate), 100 mM KCL, 2.0 mM MgCl 2 , and 2.0 mM ethylene glycol tetraacetic acid, pH 7.4) were added meat sample (1g), which was then homogenized in a Heidloph (DIAX-900,Germany). Two equal aliquots of meat homogenate (2 mL) were taken from a sample, precipitated with 2 mL of 20% trichloroacetic acid (TCA), and centrifuged at 12,000 × g for 5 min at room temperature. After centrifugation, one of the pellets was dissolved with 2 mL of 10 mM 2,4-dinitrophenylhydrazine in 2 M HCl and the other with 2 M HCl (blank), and incubated for 30 min in the dark. During incubation, samples were vortexed for 10 s every 3 min. The protein was further precipitated with 2 mL of 20% TCA and centrifuged at 12,000 xg for 5 min. The 2,4-dinitrophenylhydrazine was removed by washing the pellets 3 times with 4 mL of 10 mM HCl in 1:1 (vol/vol) ethanol:ethyl acetate, followed by centrifugation at 12,000 × g for 5 min. The pellets were finally solubilized in 2 mL of 6.0 mM guanidine hydrochloride dissolved in 20 mM potassium dihydrogen phosphate (pH = 2.3). The samples were kept at 5 °C overnight, centrifuged and their supernatant absorbance was read at 370 nm. The value of blank sample was subtracted from their corresponding sample values. The carbonyl content was calculated as nmol/mg protein using absorption coefficient of 22,000/M/cm, as described by Levine et al. (1994).

Effect of Origanum syriacum L. Essential Oil on the Storage Stability of Cooked Chicken Meat
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Sensory evaluation
Trained sensory panels were used to evaluate the sensory characteristics of the ground cooked chicken thigh meat. Sensory panels evaluated the cooked meat for: chicken aroma, oregano spice odor, oxidation odor, and overall acceptability. The meat samples submitted to the six different treatments were prepared using the same method described in the oxidation analysis section.
The meat was refrigerated at 4°C for four days before cooking and for each evaluation session. Ten trained panelists (students and staff of Muta'h University) participated in each session. The evaluations were done twice after cooling the cooked meat patties to room temperature at 25 °C. For training, 3 one-hour sessions were held using commercial and experimental products to develop descriptive terms for the desired attributes.
The cooked meat samples (10g) were evaluated by the panelists after cooling to the room temperature (25°C). The panelist was given 1 glass vial (20 mL) of each treatment to evaluate odor of cooked thigh meat samples. All samples vials were labeled by a randomly selected three-digit number. Panelists were asked to smell the samples in random order and record the intensity of odor or over all acceptability on the scale line.

Cooking loss percentage
Ground chicken fresh meat samples (100g) were prepared as the method described in lipid and protein oxidation part for all treatments. Ground meat patties were stored for 4 days in oxygen-permeable bags at 4°C, weighed and vacuum-packed before cooking. The meat was cooked at a constant temperature using pre-heated water bath (Memmert, WNB 14, Germany) to 80 °C internal temperature of for 90 min for the maximum expected water loss (Murphy & Marks, 2000). After cooking, meat samples were cooled in cold water bath using to 20°C internal temperature, then water blotted or purged until the sample became dry. Cooking loss percentage was calculated as percent weight reduction of the cooked sample compared to the raw fresh meat sample using the following equation: Cooking loss % = (raw meat weight -cooked meat weight) × 100 (raw meat weight)

Raw meat pH values
The pH value of the ground raw meat samples were determined using a pH meter (PL-600, pH/mV/Temp Meter, Taiwan) after homogenizing 1.0-g samples with 9 mL de-ionized distilled water (DDW) (Sebranek et al., 2001).

Statistical analysis
Data were analyzed using the generalized linear model procedures (Proc. GLM, SAS program, version 9.3, 2012). Mean values and standard error of the means (SEM) were reported. The significance was defined at p<0.05 and Tukey's test or Tukey's Multiple Range test were used to determine whether there are significant differences between mean values.

Lipid Oxidation
In general, TBARS values results are higher in cooked than in raw chicken meat samples (Al-Hijazeen et al., 2016a and b; Ahn et al., 2009). This due to the higher oxidation rate of cooked meat, in which antioxidant enzymes are denatured, ion is released from the intracellular compartment and the phospholipid bilayer of the cell membrane is damaged, allowing oxygen and catalysts to enter the cell during cooking and storage (Ahn & Lee, 2002). No difference (p>0.05) in TBARS values among treatments were detected on day 0 of storage for both breast and thigh meat samples (Table 1). On days 4 and 7 of storage, both breast and thigh samples containing additives showed significantly lower TBARS values (p<0.05) compared with the control samples. However, no TBARS differences among additive-treated breast samples were found (p>0.05) on days 4 and 7 of storage. On the other hand, evaluating additive-treated thigh samples, the TBARS level of samples treated with OE at 100 ppm were significantly higher than those treated with OE at 150 ppm, BHA, E-300, and E-250 on day 4 and a wide variation was observed on day 7 of storage. This may be due to the higher fat content of thigh meat compared to the breast meat samples. At the end of storage time (day 7), OE at 150 ppm and E-250 had the highest antioxidant effect on thigh meat compared to the other treatments, whereas L-ascorbic acid was the least effective additive to delay lipid oxidation in thigh samples on day 7. No statistical differences in TBARS levels (p>0.05) between E-250 and OE at 150 ppm on day 7 of storage were found. This indicates that OE at 150 ppm may have comparable effect to E-250 and other synthetic antioxidants onmalonaldehy deformation in cooked thigh meat samples (Table 1). This result agrees with other research studies where OE directly added to the meat and meat products (Chouliara et al., 2007;Fasseas et al., 2008;Kumar et al., 2015). However, further research studies are needed to investigate the partial replacement of E-250with oregano essential oil.   Values with different letters within a row are significantly different (p<0.05). n=4.
x-z-Values with different letters within a column are significantly different (p<0.05). TBARS value in mg malonaldehyde/kg meat. Treatments: Control; 100 ppm OE; 150 ppm OE; 300 ppm E-300; 5 ppm and 14 ppm BHA in the breast and thigh meat, respectively; 150 ppm E-250.

Protein oxidation
Protein oxidation significantly affects meat quality and its storage stability, and it is highly correlated with lipid oxidation (Lund et al., 2011;Eztevies, 2011;Howell et al., 2001;Ahn et al., 2009). Changes in protein functionality, such as gelation, viscosity, solubility, and emulsion stability, as a result of oxidation process affects the final quality of meat products (Xiong, 2000). In the present study, protein oxidation (total carbonyl levels) were measured and determined according to the general method described by Lund et al. (2008). No significant differences (p>0.05) were detected in total carbonyl values (nmol/mg protein) among treatments in breast and thigh samples on day 0 of storage. However, total carbonyl values were significantly reduced (p<0.05) on days4 and 7 of storage in breast and thigh additive-treated samples compared with the control samples (Table2), except for breast meat treated with OE at 100 ppm, which presented an intermediate value.

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In general, these results are in agreement with those of Al-Hijazeen et al. (2016a and b), and other researchers (Estevez et al., 2005;Sun et al., 2010), who reported that the total carbonyl values reached up to 5 nmol/mg protein in cooked chicken meat. The addition of BHA or E-300significantly reduced carbonyl values both in breast and thigh meats (p< 0.05) relative to the control treatment measured on day 7; however, among the tested additives, E-300 resulted in numerically higher total carbonyl values in thigh meat on day 7. Both breast and thigh samples treated with oregano essential oil at 150 ppm and E-250 presented numerically lower total carbonyl values, suggesting higher antioxidant effect compared with the other treatments. Although OE at 150 ppm had anti-carbonyl formation activity both in breast and thigh samples, higher total carbonyl values (nmol/ mg of protein) were detected in thigh samples. Al-Hijazeen et al. (2016a), studying the effect of different levels of Origanum vulgare addition to ground cooked breast chicken meat. In addition, both OE levels (100 & 150 ppm) had significantly reduced thigh protein oxidation during storage time. Similar effects were found by Fasseas et al. (2008) when adding OE (3%w/w) to ground cooked pork and beef.

Cooking loss and ultimate pH values
The pH value was measured in all treatment samples before cooking, and was not significantly different (p> 0.05) among treatments. Therefore, all samples had the same pH value before the cooking loss evaluation. In addition, no significant differences (p>0.05) in cooking loss % among treatments were detected (Table 3). There is a strong relationship of cooking loss with protein and lipid oxidation of raw meat, which affects meat quality (Kumar et al., 2015;Soladye et al., 2015;Aalsyng et al., 2003). The oxidation of meat protein (tissues) usually reduces meat water-holding capacity, causing dehydration (Liu et al., 2010) and further water loss (free water) during cooking (Soladye et al., 2015;Wang et al., 2009). These results indicate that the evaluated antioxidant additives did not influence meat water-holding capacity. However, very few research studies on the effect of direct the addition OE on the storage stability and quality characteristics of cooked ground meat were published: one by Al-Hijazeen et al. (2016a) with chicken meat, and another by Fasseas et al. (2008) with pork and beef. Oxidized raw meat may increase cooking loss (Liu et al., 2010), as oxidation affects meat water-holding capacity, fatty acid profile, protein functionality, cooking procedure, and volatile compound production (Lorenzo & Dominguez, 2014). Water-holding capacity of raw meat is also affected by total protein content and their amino acid composition. This agrees with the total carbonyl value differences obtained between the control and additive treatments; however, only numerically higher cooking loss values were observed (p>0.05). It should be mentioned that meat cooking loss may also affected by many other factors (internal and external), such as grinding, surface area, fat content, cooking procedure, muscle fiber type, and freshness (Murphy & Marks, 2000;Guerrero et al., 2013;Barbanti & Pasquini, 2005;Lorenzo et al., 2014).

Sensorial evaluation
The sensorial evaluation results of thigh meat samples are shown in Table 4.
The highest chicken aroma score (7.77) was obtained with the addition of E-250addition, followed by the addition of OE at 150 ppm (6.94), which, however, was not statistically different (p>0.05) from the scores obtained with BHA (6.82), E-300 (6.50), and OE at 100 ppm (5.73). The meat sample with no additives received the lowest (p<0.05) chicken aroma score, which was statistically similar to that obtained with E-300 and OE at 100 ppm.
The oregano odor was strong enough to be clearly recognized by the panelists, as shown by the significantly higher scores (p<0.05) compared with the other treatments. This is consistent with the findings of other researchers, who studied the effect of OE addition by dipping (Pavelkova et al., 2013), coating (Vital et al., 2016), and/or mixing (Al-Hijazeen et al., 2016a) on meat sensory attributes and consumer acceptability.
The high estoxidation odor score was obtained in the control samples (6.05), while the lowest in the samples added with OE at 150 ppm (3.02). The oxidation odor score obtained with OE at 100 ppm (4.34) was not statistically different from those obtained with the other treatments, whereas E-250 and BHA scores were statistically similar to the obtained with OE at 150 ppm. The low oxidation odor score obtained with OE at 150 ppm suggests it delayed oxidation and rancidity development.
The overall acceptability score of the control sample was significantly lower (p<0.05), compared with the other treatments, except for E-300, which score value was intermediate. Both E-250 and OE at 150 showed numerically higher (p>0.05) overall acceptability scores, which is consistent with the lipid and protein oxidation results obtained with these additives.

CONCLUSIONS
The addition of Origanum syriacum L. essential oil at 100 and 150 ppm to ground chicken breast and thigh meat reduced meat lipid and protein oxidation, demonstrating it was very effective to enhance the storage stability, comparable to that obtained with the evaluated synthetic meat preservativesE-250, BHA, and E-300. In addition, the thigh chicken meat with OE at 150 ppm promoted excellent sensory attribute scores. Therefore, it is concluded that OE at 150 ppm may be used in replacement of synthetic antioxidants to improve the storage stability of chicken meat.