Recommended level of rosemary extract (Rosmarinus officinalis L.) based on lipid oxidation, total volatiles, and sensory evaluation of treated cooked chicken meat

Abstract

This study was conducted to investigate the antioxidant effects of selected levels of rosemary extract (RE) on lipid oxidation, total volatiles (TV), and their sensory characteristics using chicken meat. Ground meat was prepared, including 1) Control, 2) 400 ppm (RE_L1), 3) 450 ppm RE_L2, 4) 150 ppm sodium nitrite (E-250), and 5) 14 ppm butylatedhydroxyanisole (BHA). Cooked meat patties were cooled and stored aerobically (4 °C) for 8 days. Meat patties were analyzed for lipid oxidation and TV profile at 0, 4, and 8 days of storage time. In addition, sensory attributes of cooked meat patties were evaluated for all treatments. Based on the current findings, RE_L2 showed the highest antioxidant effect (p < 0.05) regarding both thiobarbituric acid-reactive substances and TV during storage. Furthermore, RE_L2 as the superior additive, had an overall positive effect on sensory attributes. It can be concluded that RE_L2 was the most effective level that the meat processing industry can rely on.

Introduction

Several studies have been conducted to investigate the preservative properties of Rosemary extract (RE) using different meat sources (Sebranek et al., 2005; Abandansarie et al., 2019; Kaur et al., 2021). Most of these showed positive effects on meat quality, freshness, rancidity development, shelf-life, and overall consumer acceptability (Sebranek et al., 2005; Kahraman et al., 2015; Hussein et al., 2018; Liu et al., 2020). However, the results were varied, and differed in the recommended levels due to many factors, for example, genetic (plant species), cultivation season, origin (country of cultivation), storage conditions, method of evaluation, and extraction method (Al-Hijazeen and Al-Rawasheh, 2019; Nie et al., 2019). RE has been analyzed for its major polyphenols (Al-Hijazeen and Al-Rawasheh, 2019; González-Minero et al., 2020). The analysis showed several bioactive compounds (Monoterpenes, diterpenes, and polyphenols) such as carnosic acid, rosmanol, carnosol, rosmariquinone and rosmaridiphenol, caffeic acid, and ursolic acid, which are responsible for the antioxidant activity of RE (Aruoma et al., 1992; Basaga et al., 1997; Raskovic et al., 2014; Mena et al., 2016). However, carnosic acid and carnosol constituents represented 90% of the antioxidant activity of RE (Aruoma et al., 1992; Erkan et al., 2008; Kaur et al., 2021). This activity is explained by the important role of these phenols (e.g., diterpene) as hydrogen donor sources and in preventing free-radical formation during food storage (Houlihan et al., 1984; Schwarz and Terns, 1992; Hall et al., 1998; Richhelmer et al., 1999; Birtic et al., 2015).

According to a recent investigation about the antioxidant effect of RE, the recommended level for use in the preservation of different meats and foods ranged between 200 and 1 600 mg/kg (Stoick et al., 1991; Shahidi and Wanasundara, 1992; Sebranek et al., 2005; Estevez et al., 2005; Kahraman et al., 2015; Abandansarie et al., 2019; Kaur et al., 2021). In addition, the effective level of plant-derived natural antioxidants depends on many internal and external factors in the meat (e.g., storage temperature, meat composition, free iron, pH...etc.), which will affect auto-oxidation development (Ahn et al., 1998; Manessis et al., 2020). Generally, it is well known that primary (hydroperoxide-ROOH) and secondary (volatiles such as ketones, aldehydes, and hydrocarbons) lipid oxidation products are the major compounds causing meat deterioration, the development of warmed-over flavor, interaction with protein functionality, and overall consumer acceptability (Ahn et al., 1998; 2001; Domínguez et al., 2019). Researchers found a positive correlation between these compounds, which could be used to better predict meat status (Al-Hijazeen, 2016a, b; Kosowska et al., 2017; Mancinelli et al., 2021). For instance, a positive correlation between total hexanal and thiobarbituric-reactive substance (TBARS) values was found using meat samples (Ahn et al., 2000; Jo et al., 2006; Ahn et al., 2009). Based on the current meat quality findings, it is clear that a stronger conclusion can be achieved by connecting primary and secondary lipid oxidation and, finally, its relation to sensory assessment. This will aid in reaching a stronger scientific conclusion, especially for industrial uses.

The idea of using natural plant sources and their extracts to replace synthetic preservatives is of great interest and highly applicable (Kumar, 2015; Manessis et al., 2020; Lungu et al., 2020). In addition, the novel approach of both the food industry and consumers to produce more organic and natural food without using synthetic antioxidants (SA) has gained momentum rapidly (Abdel-Hamied et al., 2009; Taghvaei and Jafari, 2015; Manessis et al., 2020). Furthermore, the negative effects of adding SA as food preservatives on human health also encourage researchers to move in this direction. However, the most reasonable point affecting consumer confidence is the carcinogenic and toxicological effects of SA (Altmann et al., 1986; Abdel-Hamied et al., 2009; Kumar et al., 2015; Kaur et al., 2021). For example, Sodium nitrite (E-250) is one of the most important preservative and curing ingredients SA stabilizing and enhances meat color and their flavor (Honikel, 2008; Sindelar and Milkowski, 2011). However, the new approach now is to decrease using or finding good combination (natural plants additives) with E-250 which will decrease the amount of nitrosamine formed in the finished meat products (Honikel, 2008; Oostindjer et al., 2014). RE is classified as one of the top plant-derived antioxidants and has a high preservation effect on many food types (Al-Hijazeen and Al-Rawasheh, 2019; Manessis et al., 2020). In Jordan, rosemary is well known and cultivated very widely due to its medicinal uses (Alzoubi et al., 2014; Allawzi et al., 2019). However, few research studies have investigated its unique composition and effect on cooked ground meat quality as a natural commercial additive. Furthermore, in current study the levels of RE (cultivated in Jordan) were different from the previous research (Al-Hijazeen and Al-Rawasheh, 2019) to achieve the optimum antioxidant effects. The major goals of the current research were 1) to study the effect of adding selected RE (cultivated in Jordan) levels on lipid oxidation, total volatiles, and sensory attributes using cooked ground meat; 2) to compare this effect with that of commercial SA; and 3) to reach stronger conclusions and new recommendation levels for commercial uses depending on the progress of lipid oxidation and their volatiles.

Materials and Methods

Essential oil composition of Rosmarinus officinalis L.    Essential oil of RE (rosemary cultivated in south Jordan in the Al-Karak region) consisting of four samples (in duplicate) was measured for average phenolic diterpenes including carnosol, carnosic acid, rosmanol, and rosmarinic acid by the method of Okamura et al. (1994) at the Royal Scientific Society (RSS) Amman, Jordan. An isocratic High Performance Liquid Chromatography (HPLC) system (Shimadzu, Kyoto, Japan) was used. The chromatographic system was equipped with a controller system (CBM-20A, Shimadzu), an Auto-Sampler (SIL-20AC), a variable wavelength ultraviolet/visible detector (Model SPD-10Avp, Shimadzu), an insulated column oven (CTO-20AC, Shimadzu), and a pumping system (LC-20AD, Shimadzu).

Meat patty preparation    Meat patty preparation was conducted at Mutah University (Agriculture Collage / Department of Animal Production) according to the method of Al-Hijazeen and Al-Rawashdeh (2019). Raw meat (thigh) was obtained from broilers raised and slaughtered in the department farm facility, where all chicken had been checked and veterinary qualified as in good health (Project reference number: 120/14/115). After slaughtering, all chicken carcasses were immersed in cold water mixed with ice for one hour and kept in a cold refrigerated (4 °C) room. Lean raw (thigh) meat was prepared, vacuum packaged in oxygen impermeable bags, and stored at –18 °C after deboning and removal of all non-meat components.

At the beginning, the meat was thawed before undergoing double grinding (Type DKA1, with 8-mm and 3-mm plates, Moulinex, France) and packaging. Five different treatments were prepared, including 1) Control (without additives), 2) 400 ppm (RE_L1), 3) 450 ppm (RE_L2), 4) 150 ppm sodium nitrite (E-250), and 5) 14 ppm butylatedhydroxyanisole (BHA). However, the amount of BHA was calculated based on the fat content in the thigh chicken meat. Highly purified and steam distilled RE was purchased from a certified company in Jordan (Green Fields Factory for oils, Amman, Jordan). Powdered E-250 (Gainland Chemical Company-GCC, factory road; UK) was dissolved in distilled water (DDW), and then stock oil emulsion (water in mineral oil) was prepared to mix with the raw meat. In addition, RE and BHA were weighed, dissolved in ethanol (100%), and then mixed with mineral oil to prepare working solutions. However, the ethanol was separated from the stock solution using a rotary evaporator (Model Laborota 4001-efficient, Heidoph, Schwabach, Germany,) at 70 °C and a vacuum pressure of 175 mbar. Each antioxidant additive was mixed for 3 min with the ground meat using a bowl mixer (Model KM-331, Kenwood Limited, Havant, UK). All treatment batches had the same amount of mineral oil and water added during meat patty preparation. Individual raw meat samples of each treatment were checked for ultimate pH and proximate composition before cooking. In the cooking part, the raw meat patties were first packaged in oxygen-impermeable vacuum bags (Ehsan & Tahssin Baalbaki CO, Amman, Jordan) and then cooked in-bag at 90 °C in a water bath (WNB 14, Memmert, GMbH + Co. KG, Schwabach, Germany) to obtain an internal temperature of 75 °C. After that, all meat patties (100 g) were cooled and transferred to new oxygen-permeable bags (polyethylene, size 11 × 25 cm, Future for Plastic Industry, Al-Moumtaz bags, Co. Ltd., Amman, Jordan), and left at 4 °C to be analyzed for TBARS and total volatiles at 0, 4, and 8 days. Separate samples (cooked) were used in a similarly described protocol, which evaluated cooking loss% and specific sensory evaluation attributes. These attributes were selected on the basis of their relation to lipid oxidation (off odor-flavor development) progress. Furthermore, the raw meat patties were stored at 4 °C for 4 days before cooking and performing each sensory evaluation session.

pH of raw meat    The ultimate pH values of the fresh thigh meat samples were measured using a pH meter (PL-600, Taiwan) after homogenizing 1.0-g samples with 9 mL deionized distilled water (DDW), as described in the method of Sebranek et al. (2001).

Cooking loss percentage    The cooking loss percentage (%) of ground chicken thigh meat was measured according to the method described by Al-Hijazeen and Al-Rawashdeh (2019).

Proximate composition    Meat batch of all treatments were analyzed for their proximate composition of fat, protein, water, and average ash percentage before cooking preparation. Samples from each treatment (two sub-samples from each batch) (n = 4) were used in these analyses using standard methods (AOAC, 2000).

Thiobarbituric acid-reactive substances (TBARS) measurement    Lipid oxidation of ground meat samples was measured using the TBARS method (Ahn et al., 1998) as described by Al-Hijazeen et al. (2016 b). All chemicals, stock solutions, and equipment were prepared before starting chemical analysis. The TBARS number was reported as mg of malondialdehyde (MDA) per kg of meat.

Sensory panel evaluation    A professional team of 10 trained panelist participated in the evaluation of selected sensory attributes of ground (cooked thigh) meat as described by Al-Hijazeen and Al-Rawasheh (2019). All attributes were considered in relation to or connected with primary and secondary lipid oxidation. Aerobic-stored cooked meat was evaluated first regarding its egg-like odor, then for spice and oxidation odor, and finally, for overall acceptability. Five treatment samples were prepared as described in the lipid oxidation part, to evaluate the effect of using different levels of RE and other SA on these attributes. In this panel of the current study, meat samples were cooled and stored at 4 °C for four days before cooking and for each evaluation progress stage. Trainers from Mutah University (students and staff) participated in each session. All treated cooked meat samples were cooled to room temperature (25 °C), and the evaluation was repeated twice to decrease variability. For training, three rounds of one-hour sessions were conducted using commercial and experimental products to perform descriptive terms of these selected attributes. All characters were evaluated using a line scale without numbers (numerical value 9 units) with graduations from 0 to 9. For example, for overall acceptability of cooked meat samples, 9 represented extremely desirable, and 0 represented extremely undesirable (9 : extremely desirable, 8 : very, 7 : moderate, 6 : slightly, 5 : neither desirable nor undesirable, 4 : slightly, 3 : moderately, 2 : very, and 1 : extremely undesirable). Similar terminology (e.g., detectable or undetectable) was used for aroma (egg-like, spice, and oxidation odor) attributes. Evaluation sessions for cooked thigh meat were performed on different days to avoid any variability. The cooked meats (10 g/each) were evaluated by the panelists for each treatment after cooling to a room temperature of 25 °C. The panelist tested one glass vial (20 mL) of each treatment to evaluate odor attributes of cooked thigh meat samples. After the cooked meat samples were placed in closed vials, they were labeled with three randomly chosen numbers (3-digit codes) according to the sensory evaluation standard guidelines. All panelists were asked to smell samples randomly, record the intensity of the odor, and give their overall acceptability using scale-line sheet.

Total volatile profile of cooked meat samples    Total volatiles (hydrocarbons, aldehydes, sulfuric, ketones, etc.) of cooked thigh meat (ground) were measured according to the method of Ahn et al. (2001) using a GC-MS (QP2010nc System, Shimadzu) connected with a purge and trap concentrator (O.I.Analytical, Eclipse; Model 4660). The volatile analysis was performed at RSS by highly trained and qualified specialists. Samples of five treatments were prepared similarly to in the previous part, and then cooked meat samples (3 g/each) were placed in small vials and analyzed by GC-MS. The identification of each peak was achieved by Wiley Library, and the area of each peak was integrated. The total peak area (total ion counts × 10⁴) was expressed as an indicator of volatiles generated from cooked meat samples.

Statistical analysis    All data were analyzed using the procedures of the generalized linear model (Proc. GLM, SAS program, version 9.3, 2012). The statistical analysis was performed (Proc. GLM, SAS program, version 9.3, 2012), and mean values with their standard errors (SEM) were reported. Significance was defined at p < 0.05 and Tukey's test or Tukey's Multiple Range test were used to determine whether there were significant differences between mean values. Pearson's correlation coefficient was measured for the control treatment samples regarding TBARS results and their selected total volatiles values (TV) samples during storage.

Results and Discussion

Essential oil composition    Rosemary (Perennial shrub) is a medicinal plant of Mediterranean origin widely distributed around the world (Nieto et al., 2018). However, its oil extract content and quality are highly variable and affected by many factors, such as storage and harvesting conditions, soil, extraction methods, and genetic and seasonal effects (González-Minero et al., 2020; Andrade et al., 2018; Nieto et al., 2018). In addition, it is important to know that leaves and stems of currently tested rosemary were cultivated after the young stage of development to achieve the highest accumulation and concentration values. Mean value percentages determined by HPLC analyses of RE composition showed that it contains an average of 26 ± 3% phenolic diterpenes (carnosol, 4%; carnosic acid, 6%; rosmanol, 8%; and rosmarinic acid, 8%), which represent its major antioxidant components. This was in agreement with several research studies, which found similar percentages (Andrade et al., 2018; González-Minero et al., 2020; Nieto et al., 2018; Allawzi et al., 2019). One of the most important constituents that gives high antioxidant activity to Rosmarinus officinalis L. cultivated in Jordan is its unique content of carnosol and carnosic acid compared to the other rosemary species (Alzoubi et al., 2014; Allawzi et al., 2019). Thus, the unique composition of this oil may have novel antioxidant activity, especially in meat preservation that has not been evaluated previously. Finally, the composition stability of RE is already confirmed by the manufacture which consider important as industrial food-additive characteristics.

Ultimate pH, proximate composition, and cooking loss%    Lipid oxidation and rancidity development in meat are also affected by its composition (fat and protein) and pH value, which will indirectly affect the quantity of cooking loss. Generally, it is very important to measure these parameters before performing any further meat analysis. The results showed no significant differences (p > 0.05) among all meat treatment batches regarding ultimate pH and proximate composition. This indicated that all treated samples had the same raw material conditions; however, any variability that could occur later would be considered due to treatment effects. These results agreed with those of previous researchers who tested RE and other plant-derived antioxidants on different meat sources (Manhani et al., 2018; Al-Hijazeen and Al-Rawasheh, 2019). In addition, no significant effect (p > 0.05) difference in meat cooking loss% was apparent among treatments (Table 1). All of the above results support the use of a univariate model among all treatments to investigate RE antioxidant activity.

Table 1. Effect of preservation additives on cooking loss, pA¹, and ² U- pH values of chicken thigh meat.

TRT*			Proximate Analysis
	Cooking Loss %	Fat %	Protein %	Water %	Ash%	pH Value
Control	0.19a	6.80a	18.63a	73.66a	0.92a	6.20a
REL1	0.19a	6.83a	18.61a	73.64a	0.92a	6.23a
REL2	0.19a	6.78a	18.60a	73.71a	0.91a	6.25a
E-250	0.19a	6.81a	18.60a	73.67a	0.92a	6.24a
BHA	0.19a	6.83a	18.62a	73.64a	0.91a	6.24a
SEMc	0.006	0.126	0.115	0.164	0.016	0.022

*  Treatments: Control; REL1; REL2; E-250; Oregano; and 14 ppm BHA; n = 4.

¹  pA: Proximate analysis of fresh meat before cooking.

²  U-pH: Ultimate pH values.

Lipid oxidation    Primary lipid auto-oxidation (hydroperoxide -ROOH) is a critical process in the formation of serval secondary compounds inside the meat system. These compounds are usually the precursors of many aldehydes, hydrocarbons, and ketones, which will cause meat to have an off-odor and flavor (interaction of Maillard reaction and lipids), starting with the removal of hydrogen and finishing with active free radicals (Ahn et al., 2009; Kumar et al., 2015; Manessis et al., 2020). Many volatiles are considered good indicators and strong signs of rancidity development in meat (Kosowska et al., 2017; Bak and Richards, 2021). Among all treatments, no significant differences (p > 0.05) appeared regarding TBARS values at day 0 of storage (Table 2).

Table 2. *TBARS values of cooked chicken thigh meat at different storage times at 4 °C.

Time	Control	REL1	REL2	E-250	BHA	SEM
		---------- TBARS (mg/kg) meat ----------
Day 0	0.966az	0.949az	0.973ay	0.987az	0.945az	0.053
Day 4	3.427ay	1.262bcy	1.115cy	1.246bcy	1.417by	0.040
Day 8	7.094ax	2.947bx	2.115cx	2.800bx	3.164bx	0.105
SEM	0.119	0.043	0.061	0.045	0.065

^x-z-Values with different letters within a column are significantly different (p < 0.05).

*  TBARS value in mg malonaldehyde/kg meat.

However, all supplements showed significant (P < 0.05) antioxidant effects, delaying lipid oxidation compared to the control at day 4. Generally, both RE levels (400-450 ppm) and E-250 showed greater effects than BHA addition during storage. The highest antioxidant effect at day 4 was found in meat samples treated with 450 ppm of RE. However, the antioxidant activity of RE was well explained by the composition of phenolic compounds (Nieto et al., 2018; Abandansarie et al., 2019; Sierżant et al., 2021). These compounds decrease lipid oxidation through several mechanisms, such as decreasing the ability of lipids to donate their hydrogen and delaying any further free radical formation (Kumar et al., 2015; Nieto et al., 2018). In addition, RE has potential antioxidant activity as a consequence of the synergistic effect of its bioactive compounds in preventing and delaying reactive oxygen species (ROS) formation (González-Minero et al., 2020). At the end of the storage time (day 8) RE_L2 showed a significantly greater (p < 0.05) effect than the other additives (RE_L1, E-250, BHA). In addition, BHA addition showed the lowest effect among all additives, with no significant differences (p > 0.05) at day 8 compared with RE_L1, and E-250. Finally, the antioxidant effect of RE_L1 and E-250 was very close and comparable throughout the storage time. Thus, RE_L2 could be the best natural replacement that may decrease secondary volatile formation.

Total volatiles    In lipid (auto-oxidation) oxidation of cooked meat, hundreds of unique volatiles formed during the storage period (Du et al., 2003; Kosowska et al., 2017). Volatiles such as aldehydes, ketones, hydrocarbons, carboxylic acids, and esters are correlated well with meat off-odor/flavor and rancidity development, which is clarified in Table 7 and was reported previously (Du et al., 2003; Nam et al., 2003). All treatments evaluated by GC-MS showed similar quantitative and qualitative volatile profiles at day 0 of the storage period (Table 3).

Table 3. Volatiles of cooked thigh meat treated with different antioxidants at day 0.

	Total ion counts * 104
Compounds	CONT	REL1	REL2	E-250	BHA	SEM
Pentane	4218a	2998b	2468b	2642b	3289ab	247
Carbon disulfide	5430a	4024a	4115a	5016a	5529a	1077
2-Propanone	3552a	3332a	2938a	3304a	3606a	182.40
1-Pentene	398a	357a	360a	463a	225a	67.59
Hexane	16365a	13216a	12203a	13104a	15895a	1082.06
Butanal	2442a	2025a	1936a	2327a	2042a	221
Dimethyl disulfide	4531a	4634a	4223a	3945a	4478a	432.75
Octane	1982a	1792a	1635a	2063a	2276a	162.55
Propanal	3287a	3177a	2215a	2902a	3120a	359.12
Hexanal	30539a	28596a	28563a	28542a	28734a	565.29
Pentanal	4378a	4160a	3644a	3934a	4361a	258.30
Nonanal	3502a	2608a	2549a	2489a	3204a	281
α-Pinene	0b	156b	193a	0b	0b	6.81
Camphene	0b	300a	358a	0b	0b	32.42
Limonene	0b	137a	140a	0b	0b	7.96
1.8-cineole	0c	140b	230a	0c	0c	5.58
α –Terpineol	0b	96a	125a	0b	0b	8.44
Camphor	0c	36b	71a	0c	0c	4.74

Different letters (a-d) within a row indicate significantly different values (p < 0.05), n = 4.

Treatments: Control, RE_L1; RE_L2; E-250; 14 ppm BHA.

In addition, no significant differences (p > 0.05) among all treatments were shown, and a small numerical effect was found for RE and E-250. This could be due to their antioxidant effects on fresh meat before cooking. In addition, a minor amount of certain phenolic monoterpenes was detected (α-pinene, camphene, limonene, 1,8-cineole, and camphor) formed by RE vials at both levels. These volatiles may have significant effects on the overall meat odor and flavor. However, the highest amount of these volatiles appeared in RE_L2 vials, which reflects their oil concentration and composition. At day 4, significant differences (p < 0.05) and the variation among treatments became apparent and were maximized for most volatiles, especially those correlated with lipid oxidation. Ketones, aldehydes (nonanal, hexanal, butanal, etc.) some sulfuric compounds (e.g., carbon disulfide) were significantly (p < 0.05) lower in all treated samples compared to the control treatment at day 4. This variation among treatments increased over time due to the formation of additional free radicals as primary lipid oxidation progressed. Researchers reported a good correlation between aldehyde formation and results of the TBARS test (Du et al., 2003). Hexanal was one of those volatiles reported as an excellent indicator of oxidation odor and flavor development (Al-Hijazeen et al., 2016a; Bak and Richards, 2021). Some sulfuric compounds (in relation to an egg-like odor) were decreased after day 4 of the storage period for all treatment vials. This was in agreement with several research studies evaluating the volatile profiles of cooked meat (Lee et al., 2003; Al-Hijazeen et al., 2016a, b).

However, GC-MS analysis of raw and cooked chicken meat showed that sulfuric volatiles in aerobically stored meats escaped from their vials due to their high volatility (Nam et al., 2002; Nam et al., 2003). Among all additives, there were no significant differences (p > 0.05) at day 8 in most volatiles; however, RE_L2 showed the greatest numerical effect, delaying off-odor volatile formation. For example, aldehydes (hexanal, pentanal, and nonanal) were formed in the lowest amounts in the vials of meat samples treated with RE_L2. During the storage period, the antioxidant effects of BHA and RE_L1 were comparable. Minor volatiles, such as ethyl propionate, 2-butanone, ethyl acetate, and S-methyl ethane appeared inconsistently and are not shown in the final tables. In addition, the main diterpenes (carnosic acid, carnosol, rosmarol, epirosmanol, and isorosmanol) were not detected clearly by GC-MS for many reasons, such as their interaction with the meat system and low volatility compared to other terpenes found in RE.

Table 4. Volatiles in cooked thigh meat patties treated with different antioxidants at day 4.

	Total ion counts * 104
Compounds	CONT	REL1	REL2	E-250	BHA	SEM
Pentane	5720a	5410a	5081a	5051a	5484a	239.79
Carbon disulfide	3762a	2687ab	2473b	2130b	2951ab	249.84
2-Propanone	12691a	9798ab	7880b	8919ab	9163ab	938.80
1-Pentene	1112a	864a	792a	698a	784a	171.95
Hexane	17844a	15217ab	13250b	13270b	13516b	668.55
Butanal	5529a	4310ab	4035b	3926b	4497ab	311.50
Dimethyl disulfide	6423a	4442a	4230a	4304a	5506a	676.37
Octane	3233a	3056a	2719a	2765a	2877a	420.45
Propanal	8497a	8153a	5016b	6195ab	8295a	603.17
Hexanal	40886a	37073ab	33171b	33330b	37272ab	1233.17
Pentanal	6260a	5655a	4577a	5164a	5507a	545.34
Nonanal	4158a	3314b	2511c	2846bc	3404b	166.78
α-Pinene	0	0	0	0	0	0
Camphene	0b	297a	322a	0b	0b	20.82
Limonene	0b	65ab	131a	0b	0b	19.88
1.8-cineole	0c	145b	225a	0c	0c	3.41
α–Terpineol	0b	101a	125a	0b	0b	11.31
Camphor	0b	46ab	112a	0b	0b	15.86

Different letters (a–d) within a row indicate significantly different values (p < 0.05), n = 4.

Treatments: Control, RE_L1; RE_L2; E-250; 14 ppm BHA.

Table 5. Volatiles in cooked thigh meat patties treated with different antioxidants at day 8.

	Total ion counts*104
Compound	CONT	REL1	REL2	E-250	BHA	SEM
Pentane	9944a	7931a	7034a	7040a	7234a	1184.89
Carbon disulfide	2529a	2106b	2013b	2025b	2230ab	86.15
2-Propanone	20606a	19751a	17951a	18085a	19369a	1190.20
1-Pentene	3460a	2751a	2737a	2325a	2514a	343.92
Hexane	31032a	28665ab	27753ab	26133b	28201ab	901.71
Butanal	13276a	10820ab	9429b	8375b	10070b	721.07
Dimethyl disulfide	5793a	4812ab	3753ab	3330b	4579ab	528.28
Octane	9553a	8632a	8263a	8202a	8351a	1488
Propanal	14830a	12686ab	11914b	11013b	12938ab	501.72
Hexanal	61393a	55973b	52017b	54314b	56249ab	1188.72
Pentanal	10210a	7373ab	6658b	6807b	7513ab	697.16
Nonanal	6537a	4524ab	3889b	4210b	4606ab	496.11
α-Pinene	0	0	0	0	0	0
Camphene	0b	298a	300a	0b	0b	9.26
Limonene	0c	56b	130a	0c	0c	4.475
1.8-cineole	0c	135b	225a	0c	0c	5.66
α–Terpineol	0b	90a	100a	0b	0b	5.86
Camphor	0b	73a	112a	0b	0b	9.01

Different letters (a–d) within a row indicate significantly different values (p < 0.05), n = 4.

Treatments: Control; RE_L1; RE_L2; E-250; 14 ppm BHA.

Sensory panel assessment    The effects of RE addition on the sensory characteristics of different meat types are well studied (Thongtan et al., 2005: Feng et al., 2016; Manhani et al., 2018; Szymandera-Buszka et al., 2020). Most of these had positive effects, as indicated by higher scores given by the sensory panel (Estevez et al., 2005; Zhang et al., 2016; Hussein et al., 2018). In this study, all treatments showed a significant (p < 0.05) antioxidant impact regarding egg-like and oxidation odor attributes (Table 6).

Table 6. Sensory attribute means of cooked thigh meat patties.

	Sensory attributesb
TRT*	Egg like Odor	Spice Odor	Oxidation Odor	Over All Acceptability
Control	7.56a	0.73c	7.23a	3.42c
REL1	5.12bc	6.14b	4.55bc	6.73ab
REL2	4.80c	7.19a	3.69c	7.47a
E-250	5.25bc	0.70c	4.45bc	6.78ab
BHA	5.88b	0.72c	5.14b	5.72b
SEMc	0.266	0.162	0.244	0.325

*  Treatments: Control; RE_L1; RE_L2; E-250; 14 ppm BHA.

^b  Sensory attributes: Samples were evaluated on day 4.

SEM^c: Standard error of the mean.

^a-f  Mean within the same column with different superscripts are different (p < 0.05), n = 10.

Table 7. Pearson correlation coefficients between volatiles and TBARS values of control samples during storage time.

Volatiles	PCC1	Mean	STD2
2-Propanone	0.95755*	12283	7514
1-Pentene	0.94981*	1657	1424
Hexane	0.93645*	21747	7012
Butanal	0.97357*	7082	4899
Hexanal	0.98948*	44272	13560
Pentanal	0.94233*	6949	2658
Nonanal	0.94189*	4732	1434
Carbon disulfide	(-) 0.88269*	3907	1371
Dimethyl disulfide	0.28763	5582	1501

**p < 0.01: Highly significant difference.

*  0.01 < p < 0.05: Strong significant difference.

¶ 0.05 < p < 0.1: Low significant difference.

¹  PCC: Pearson correlation coefficients, n = 12, Prob > | r | under HO: Rho = 0.

²  STD: Stander deviation.

However, RE_L2 (450 ppm) showed the greatest ability to decrease the formation volatiles, which are responsible for the egg-like odor (e.g., sulfuric compounds: carbon disulfide, dimethyl disulfide, etc.), compared to other additives. The formation of these secondary volatiles (aldehydes, hydrocarbons, ketones, etc.) usually increased during the storage period and participated more in the development of a rancid odor (Du et al., 2003; Ahn et al., 2009; Bak and Richards, 2021). However, BHA showed the lowest effect (higher values) on egg-like odor formation, compared to other additives. In addition, RE and E-250 additives were associated with the greatest decreases in oxidation odor score values. There were no significant differences (p > 0.05) regarding oxidation odor among RE_L1&2 and E-250. In addition, Both RE_L2 and E-250 showed the highest overall acceptability score values compared to the other treatments. The extension of meat shelf life by RE and its positive sensorial effect was recently explained and discussed thoroughly (Resendiz-Cruz et al., 2021; Bak and Richards, 2021). The main factor responsible for this ability was its unique phenolic antioxidant constituents (Feng et al., 2016; Hussein et al., 2018; Nieto et al., 2018; Resendiz-Cruz et al., 2021). Antioxidant effects are linked to the improvement achieved by decreasing primary and secondary compounds formed during lipid oxidation. Generally, panelists prefer RE in terms of sensorial aspects, even though some other natural additives may have higher antioxidant effects (Al-Hijazeen and Al-Rawasheh, 2019; Szymandera-Buszka et al., 2020). Correlation analysis (Table 7) confirmed this relationship. Finally, the current sensory evaluation showed that the concentration of RE in RE_L2 could be recommend for industrial uses.

Conclusion

Direct addition of Rosmarinus officinalis L. at both levels exhibited positive effects on meat quality and decreased rancidity development. All additives showed significant (p < 0.05) antioxidant effects regarding lipid oxidation (TBARS) and total volatiles in cooked chicken meat. However, RE_L2 showed comparable effects to the synthetic antioxidant E-250 and higher ones than BHA. The panelists also evaluated RE_L2 as the best additive affecting the overall acceptability of meat samples. Based on all findings, 450 ppm of RE could be a suitable level recommended as a commercial replacement in cooked chicken meat products. ext,

Acknowledgements    This study was supported financially by the Deanship of Scientific Research at Mutah University, Al-karak, Jordan. Grant number: 120/14/118. The Authors would like to thank Dr. Ghaid Al-Rabadi for his expert advice regarding chicken and diet formulation.

Conflict of interest    There are no conflicts of interest to declare.

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