Effect of rosemary essential oil as nitrite substitute on quality of sausage produced using chicken fed by thymus essential oil and rapeseed oil

Abstract

The objective was to determine the effect of rosemary essential oil (REO) as nitrite substitute on the physicochemical, microbiological and sensory quality of sausage from broilers fed different levels of thymus essential oil (TEO) and rapeseed oil (RO). Broiler chicks fed TEO (0, 300 and 500 ppm) and RO (1, 3 and 5%) in a 3 × 3 factorial design. Sausages were made from meat of chicken fed TEO (0 and 500 ppm) treated with REO (120 ppm), REO:nitrite (60:60 ppm), nitrite (120 ppm) or without REO and nitrite in a 2 × 4 factorial design. Results showed that TEO increased water holding capacity and pH value and decreased malondialdehyde concentration. RO (1 and 3%) × TEO (500 ppm) group had higher unsaturated to saturated fatty acids ratio in the meat. Results showed that treatments of REO (120 ppm) decreased lipid oxidation and increased pH value of meat. Sausage produced by nitrite (120 ppm) or REO:nitrite (60:60 ppm) had lower total microbial count, lightness but higher redness and yellowness. Based on the results, TEO in chicken feed decreased lipid oxidation of sausages. Furthermore, substitution at least 60 ppm nitrite with REO had more preserving effect on quality of produced sausages.

Introduction

Chicken meat is one of widely consumed essential sources of proteins in human diets of world. Meat and meat products are essential sources of proteins in human diets. Relationship between food and human health created a concept of functional food market (Siro et al. 2008) and has promoted trying to make a sausage from chicken meat.

Lipid oxidation has negative effects on meat quality and often determines the shelf life of meat and meat products (Brenes et al. 2008). Scientific studies have indicated that the n-3 PUFA concentration of poultry products may be increased by the addition of fish oil, flax seed, linseed oil and marine algae (Abbasi et al. 2020; Basmacioglu et al. 2004). However, an increase in the amount of n-3 PUFAs in foods, especially docosahexaenoic (DHA) and eicosapentaenoic (EPA), may confer greater susceptibility to lipid oxidation, and oxidative deterioration adversely affects the sensory quality of products, including odours or flavours during storage (Gonzalez-Esquerra and Leeson 2001). However, suppression of lipid oxidation and inhibition of bacterial growth are major ways in extending the shelf life of meat and meat products (Rahiminiat et al. 2017; Mahmoodi Bardzardi et al. 2014).

Antimicrobial feed additives have been used for more than 50 years to enhance growth performance and to prevent disease in livestock feeding environments. However, the current trend is to look for alternatives because of residues in animal products and risk of resistant bacteria. Furthermore, over the last decade’s sodium nitrate and nitrite have been exploited in meat products as inhibition of microorganisms, especially Clostridium botulinum, colour and flavour development of meat products (Honikel 2008). Moreover, it can delay lipid oxidation of meat products via formation of a stable complex with Fe(II) (Honikel 2008). However, since the 1960s, concerns were raised about formation of nitrosamine compounds in meat products may cause some complications and cancers in consumers (Ferguson et al. 2004). Hence, markets have been investigated on natural ingredients and proposed plant essential oils as anti-oxidative and anti-microbial agents with minimal change in the sensory properties in diets, meat and meat products (Riel et al. 2017). Several researches showed that polyphenols, flavonoids and terpenoids obtained from different plants and vegetables could be used as nitrite replacer with strong antioxidant activity in processed meat (De Oliveira et al. 2012; Sojic et al. 2018).

Thyme (Thymus vulgaris), containing main volatile oils of thymol, p-cymene and carvacrol can be adopted as a natural alternative to antibiotics in poultry production. Rosemary (Rosmarinus officinalis L.), containing main oils of p-cymene, linalool, γ-terpinene and thymol were set as desirable antioxidant and antimicrobial agent in meat products (Turek et al. 2000).

Nevertheless, due to the multiple preservative roles of nitrite in meat products, many researches have been dealing with a strategy for total or partial substitution of nitrite by natural additives. So, the aim of this paper was to:

1. Evaluate the effect of different levels of thymus essential oil (TEO) and rapeseed oil (RO) in feeds of broilers on oxidative changes in fats of meat.

2. Investigate the effect of substitution of nitrite by rosemary essential oil (REO) on the physicochemical, microbiological and sensory quality of sausage from broilers fed TEO and RO.

Materials and methods

Experimental design and treatments

All experimental procedures used were approved by the Animal Welfare Committee of the Department of Animal and Poultry Sciences, University of Tehran. All husbandry practices and euthanasia were conducted with full consideration of animal welfare.

Five hundred and forty 1-day-old male broiler chicks (Ross 308; Mahan Breeder Company, Varamin, Tehran, Iran), were weighed and randomly assigned into nine dietary treatment groups as: 1% rapeseed oil (RO1), 3% rapeseed oil (RO3) or 5% rapeseed oil (RO5) supplemented with 3 levels of thyme essential oil (TEO) as: 0, 300, 500 ppm. Each of group was divided into five equal replicates of 12 male birds per replicate. The basal diet was formulated to meet the nutrient requirements of the broiler chickens as recommended by Ross 308 broiler management guide. The experimental diets were based on corn–soybean meal containing vegetable oil. The thymus essential oil was mixed with a carrier (rapeseed oil), which was then added to the basal diet. Feed and water were provided ad libitum. Temperature and relative humidity was maintained within the optimum range. The birds were kept in 40 Cage (1 × 1.1 m) and photoperiod of 24 h light/day was maintained during on days 1–3 of age and 23 h light/1 h of darkness on day 42. All chicks were fed starter diets (1–10 days of age), grower diets (11–24 days of age) and finisher diets (25–42 days of age).

At the 42nd day of age, immediately after the slaughtering process, bones and skin were removed from poultry meat. Chicken meats were taken directly to the production line and were packed into polyethylene bags (1 kg) and stored in temperature-controlled chambers, following the same procedures adopted by the industry. Samples were separated into eight different treatments: sausages were made from meat of chicken fed TEO (0 and 500 ppm) and RO (5%) treated with REO (120 ppm), REO:nitrite (60:60 ppm), nitrite (120 ppm) or without REO and nitrite in a 2 × 4 factorial design.

At appropriate intervals of time, random sausages samples were removed from the chambers, thawed in a refrigerator at 4 °C and submitted to lipid oxidation, pH and color parameters, proximate composition, microbiological analysis and sensory evaluation, following the methodologies described below.

Chicken meat quality measurements

Muscle pH

Breast and thigh muscles immediately and 4 h after slaughtering were measured for tissue pH at a depth of 2.5 cm using pH meter (Merohm Company, 827 Model pH lab) with a glass electrode calibrated with the phosphate buffers 4.0 and 7.0 at room temperature (21 °C) for all samples.

Water holding capacity

Water holding capacity (WHC) of the meat samples was determined based on method described by Bertram et al. (2001). About 4 h after slaughtering, 1 g of breast and thigh meats per chicken were centrifuged (4000×g for 15 min) for expressed juice determination. The water remaining after centrifugation was quantified by drying the samples at 70 °C overnight. WHC was calculated as: (weight after centrifugation − weight after drying)/initial weight × 100.

Fatty acid profile

For the analysis of fatty acid profile, total lipids of thigh meat were extracted according to procedure described by Folch et al. (1957) and extracted fat stored at − 20 °C for fatty acid analysis. The FA-methyl esters were performed based on the procedure of Metcalfe et al. (1966). The FA-methyl esters were isolated by gas chromatography on a Hewlett-Packard 5890 gas chromatograph. Fatty acid composition was determined by gas–liquid chromatography [(Perkin Elmer (Auto System) Gas Chrom, column: SGE (BPX 70 GC column) (60 m-capillary; 0.25 μm film thickness; 0.25-mm diameter WCOT fused-silica), detector: flame ionization detector (FID), column temp: ramp 1:165 °C (increase with 1 °C/min) (2 min isotherm), ramp 2:225 °C (increase with 2 °C/min) (30 min isotherm), detector temp: 250 °C, injector temp: 250 °C, elution time: 35 min, inject. amount: 2 μL, split ratio:50:1, carrier gas: Helium (He), flow rate: 1.5 mL/min]. Individual fatty acid was expressed as a percentage of total fatty acids.

Lipid oxidation

Malondialdehyde (MDA) was determined 3 days after storage in refrigerator (4 °C) by a selective third-order derivative spectrophotometric method (Botsoglou et al. 2002). Third-order derivative spectra were established by a digital differentiation of the normal spectra, using a derivative wavelength difference setting of 21 nm. The standard calibration curve was prepared using 1,1,3,3-tetraethoxypropane (Sigma Chemical Co, St. Louis, MO).

Sausage preparation

For production of poultry sausages, meat of broiler chicken (n = 40) fed TEO at levels of 0 and 500 ppm and RO (5%) in feed were used. The sausages were manufactured in Arvin Products Co. (Isfahan, Iran) according to a factory formulation including modified deboned chicken meat (70%), soy protein isolate (1%, Kron Co. China), soybean oil (7%), ice (16%), salt (1.5%), starch (3%, Glocuzan Co, Iran), sodium poly-phosphate (0.4%, Bodenhaym Co, Germany), ascorbic acid (0.5%, Bodenhaym Co, Germany), spice (0.9% including garlic, white egg, dextrose, wheat flour and stabilizer, Golha Co, Iran). Chicken meat, salt and phosphate were placed in a chilled cutter and mixed for 1 min. Subsequently, 50% of ice and spice were added and mixed. After complete homogenization, remaining 50% of ice as well as starch, ascorbic acid and oil were transferred to a cutter and the following preservatives (nitrite or rosemary essential oil) at different levels: nitrite (120 ppm) or nitrite: rosemary essential oil (60:60 ppm) or rosemary essential oil (120 ppm) or 0 ppm rosemary essential oil and nitrite were supplemented until the temperature of the mixture reached 13 °C. After the formation of an emulsion, the mixture was stuffed (Handtmann VF-200, Biberach/Riss, Germany) into polyamide casing (20 mm diameter) with approximately 0.1 kg of product per package. The sausages were cooked (Vemag, Verden, Germany) for 30 min until the internal temperature to reach 72 °C. After cooking, the sausages were cooled with cold water and stored cooked sausages (4 ± 1 °C) were analyzed at 0, 7, 14, 21, 28 and 35 days.

Sausage proximate composition

Moisture content (%), crude protein (%), and crude ash (%) were determined using the AOAC method (2000). Crude fat (%) was analyzed using the method described in Folch et al. (1957).

TBARS, sausage pH and color parameters

Lipid oxidation was determined based on TBARS method described by Raharjo et al. (1992). The absorbance of an aliquot of the solution was read at 532 nm with a UV–visible spectrophotometer (Shimadzu, Model UV-160A, Tokyo, Japan). The TBARS levels were calculated in units of mg of malonaldehyde per kg of sample. For pH determination, 5 g of sample was blended with 50 mL deionized water for 2 min and the pH of resultant suspension was measured using Crison pH meter (507Model, Crison, Barcelona, Spain) equipped with a Crison combination electrode (Cat. nr 52, Crison, Barcelona, Spain). Measurement of sausages color was performed using a calibrated Hunter Lab Colorimeter (Hunter Associates Color Quest II Laboratory, Inc., Reston, VA) at a standard observer angel of 10° with the D65 illuminant containing 20-mm port. RSEX and CIELAB mode color system were used that in which L*, a* and b* values indicated the lightness, redness and yellowness, respectively.

Sausage microbiological analysis

Sausages were aseptically removed from the bags and 10 g of each sample homogenized with 90 ml of (0.1% w/v) sterile peptone water with 0.85% (w/v) NaCl in a stomacher blender for 1 min. One milliliter of prepared appropriate decimal dilutions were pour-plated on plate count agar (PCA) for count of mesophilic aerobic bacteria and incubated at 30 °C for 48 h (Riazi et al. 2016).

Sausage sensory evaluation

Sensory evaluation was approved by the Ethics in Research Committee of the University of Tehran, Aburaihan campus (Protocol No. 281/2010). To determine the sensory quality quantitative descriptive analysis (QDA) (ISO, 13299.2 1998) was used with an unstructured, linear graphical scale; 100 mm was converted to numerical values (0–10 conventional units—c.u.). Descriptions were chosen and defined during a panel discussion and then verified in a preliminary session. Finally, color, taste, acceptability index, firmness and odor of sausage samples were evaluated using a five-point scale in which a score of 1 was bad or slightly extreme and 5 was good or much extreme.

The sensory assessment panel composed by 100 untrained panelists recruited among students, faculty and the staff in the campus. During the evaluation, the panelists were placed in private booths under incandescent/fluorescent light, with an intensity of approximately 350 lx. All samples were randomly coded using three-digit numbers and then presented to the panelists. The sausages were individually wrapped in aluminum foil and cooked in an oven with the temperature of 150 °C until the internal temperature of about 75 °C was obtained and then they were kept at about 72 °C until consumption. Then sausages were cut into rectangular pieces, approximately 1.5 × 2 cm and served (ASTM 1988). Each panelist evaluated 3 replicates of all treatment groups. Water and unsalted crackers were provided between samples as palate cleansers to remove any residual flavors (Jin et al. 2014).

Statistical analyses

Data were analyzed by the General Linear Models (GLM) of SAS (SAS 2003; Institute Inc., Cary, NC, USA). In broiler production, the factorial model (3 × 3) was included the effects of thymus essential oil, rapeseed oil levels and interaction of treatments as a completely randomized design (CRD). In sausage production, the factorial model (2 × 4) was included the effects of rosemary essential oil, sources of chicken meat and interaction of treatments as CRD. The data of sensory properties were analyzed by using non-parametric test (Kruskal–Wallis test). Analytical model and its components were:

$$Yij=\mu +Ai+Bj+\left(A+B\right)ij+eij$$

where Yij, dependent variable; μ, mean of the population; Ai, effect of treatment i; Bj, effect of treatment j; (A × B)ij = 2 way interaction of treatment i × j and eij, unexplained residual with normal distribution. The significance level was declared at P ≤ 0·05, and tend toward significance was considered at 0·05 < P ≤ 0·1 by Tukey test (SAS Institute 2003).

Results and discussion

Meat quality measurements

Water holding capacity, pH and lipid oxidation of meats

Results showed that pH values of breast and thigh muscles did not affected by RO and interaction of RO and TEO (Table 1). Nevertheless, pH values were affected (P < 0.01) by different levels of TEO. Broilers fed TEO500 had greater (P < 0.01) pH value of thigh muscle compared with TEO0, while, pH values of breast muscle increased (P < 0.01) linearly so that broilers grouped as TEO500 had the highest pH value, followed by TEO300 than TEO0 4 h after slaughter. TEO500 increased (P < 0.01) WHC value of thigh and breast muscles of birds compared to TEO0 and TEO300 had intermediate value (Table 1). In line with Qiao et al. (2001) demonstrated that low pH meat had lower WHC, leading to drip loss and cook-loss. It is well known that water holding capacity is related to pH value so that the lower final pH leads to loss of water holding capacity (Qiao et al. 2001). A rapid pH decrease may cause protein denaturation, leading to decreased juiciness and intense muscle coloration (Qiao et al. 2001).

Table 1.

Effects of different levels of thymus essential oil and rapeseed oil on lipid oxidation (all data points are mean malondialdehyde (MDA) concentrations), water holding capacity (WHC) and pH of thigh and breast meats in broiler chickens

Items	Thigh (mg MDA/kg meat)	Breast (mg MDA/kg meat)	WHC		pH in thigh muscle		pH in breast muscle
	3 days	3 days	Thigh	Breast	After slaughter	At 4 h after slaughter	After slaughter	At 4 h after slaughter
Main effects
RO levels
1%	0.094	0.053	56.68	53.60	6.64	6.28	6.7	6.17
3%	0.132	0.084	55.00	52.36	6.60	6.21	6.65	6.04
5%	0.154	0.089	54.92	51.06	6.56	6.14	6.57	5.99
SEM	0.018	0.013	1.85	1.5	0.111	0.07	0.079	0.075
TEO levels
0 ppm	0.181a	0.113a	51.72b	49.15b	6.32b	6.04b	6.40b	5.75c
300 ppm	0.139a	0.064b	54.89ab	51.81ab	6.64ab	6.23ab	6.67a	6.04b
500 ppm	0.061b	0.048b	59.98a	56.05a	6.85a	6.36a	6.85a	6.40a
SEM	0.018	0.013	1.85	1.50	0.111	0.07	0.079	0.075
RO levels × TEO levels
1%
0 ppm	0.025	0.116	53.28	49.3	6.26	6.20	6.44	5.72
300 ppm	0.102	0.085	51.33	50.30	6.64	6.38	6.81	6.26
500 ppm	0.057	0.137	50.56	47.85	7.01	6.28	6.85	6.52
3%
0 ppm	0.103	0.028	59.95	53.86	6.49	6.05	6.42	5.89
300 ppm	0.134	0.097	51.82	49.84	6.64	6.30	6.66	5.90
500 ppm	0.18	0.068	52.90	51.73	6.68	6.28	6.88	6.32
5%
0 ppm	0.156	0.015	56.79	57.64	6.20	5.86	6.34	5.66
300 ppm	0.160	0.069	61.86	56.94	6.63	6.02	6.55	5.97
500 ppm	0.226	0.061	61.29	53.59	6.87	6.54	6.83	6.35
SEM	0.032	0.023	3.22	2.60	0.192	0.121	0.136	0.131
P value
RO	0.09	0.14	0.75	0.50	0.90	0.36	0.50	0.25
TEO	0.0009	0.008	0.018	0.014	0.016	0.014	0.002	< 0.0001
RO × TEO	0.50	0.23	0.32	0.80	0.64	0.09	0.92	0.40

SEM standard error of mean, TEO thymus essential oil, RO rapeseed oil, MDA malondialdehyde, WHC water holding capacity

^a–cMeans with no common superscript within each column are significantly (P < 0.05) different

As shown in Table 1, different levels of RO and interaction of RO and TEO had no effect on MDA concentration. Nevertheless, tendency for increase (P = 0.09) in MDA concentration of thigh muscle by increasing in RO percentage of diet was noted. After 3 days of storage, broiler fed TEO500 had lower (P < 0.01) MDA concentration of thigh muscle than TE0300 and TEO0, while, MDA concentration of breast muscle was lower (P < 0.01) in TEO500 and TE0300 than TEO0. The enhancements in the general health status of the broilers fed different TEO supplementations could be attributed to pro-oxidant scavenger and anti-oxidant effects (Hady et al. 2016). Rich PUFA poultry meat is more impressionable to lipid oxidation than pork or beef, which may even develop toxic products. Marcincak et al. (2005) reported that addition of vitamin E (40 ppm) with rosemary powder (500 ppm) to feed mixes increased the oxidative stability and decreased MDA of lipids in meat 9 days after storage as comparison to the control group. In addition, it is well demonstrated that thyme oil may potent to decrease the peroxidation biomarkers in plasma and muscle and increase antioxidant abilities (Mansoub and Nezhady 2011). In line with Basmacioglu et al. (2004) reported that thyme oil, rosemary and alpha-tocopherol acetate reduced MDA concentration in poultry meat in early days of storage, while rosemary and thyme oil were more effective in reducing MDA level over a longer period of storage. Nevertheless, Attia et al. (2017) found under hot climate, serum MDA was not affected by different levels of TEO in male broiler's feed.

Fatty acids composition

As results shown in Table 2, 5% dietary supplementation of RO decreased SFA (P < 0.01) and increased UFA (P < 0.01) content of thigh muscle than 3% or 1% of RO. Additionally, UFA/SFA ratio of thigh muscle increased (P < 0.001) by increasing in RO percentage, such that RO5 had greater UFA/SFA ratio than RO3 and RO1. In this case, previously, Stanacev et al. (2014) found that replacing 4% of soybean oil with rapeseed oil in the broiler's diet reduced the percentage SFA and increased the MUFA as well as PUFA in abdominal fat pad. Also in accordance with the fatty acid composition of the oil, decreasing in proportion of linoleic acid was replaced by increasing in linolenic acid in abdominal fat. In agreement, our results showed that 5% of RO decreased palmitic and myristic acid and increased oleic and linolenic acid than 3 and 1% of RO. Jankowski et al. (2012) revealed that diet containing rapeseed oil (33.0 and 7.96% of n-6 and n-3 PUFA) had lower share of SFA and higher UFA than soybean oil. In line with Jankowski et al. (2012), our results showed that replaced rapeseed oil increased UFA in thigh muscle, by increasing in the concentrations of MUFA-C18 (oleic acid). The TEO supplementation and interaction of RO and TEO changed SFA proportion, so that the broilers fed by dietary TEO 500 and TEO300 had lower C14:0 than diet free TEO, while C16:0 fatty acid decreased by TEO 300 than TEO0 (P < 0.01) in thigh muscle in broiler chicken. It is well demonstrated that fatty acid profile of meat could be changed by adding plant oils to the feed of broilers (Zelenka et al. 2008). However, contradictory results of TEO were reported by Botsoglou et al. (2002) who claimed that alpha-tocopherol acetate may be more efficient antioxidant in some cases in broiler's feed. Based on the results showed in Table 2, interactions of RO1 and TEO500, RO5 and TEO500 as well as RO3 (TEO300 and TEO500) were noted for higher (P < 0.01) UFA/SFA ratio than TEO free diets. In line with Bolukbasi et al. (2006) maintained that addition of thyme oil to the broiler feed led to a significant decline in SFA and increase in PUFA concentrations of the breast and thigh tissues, because of antioxidant potential and impede free radical formation.

Table 2.

Effects of different levels of thymus essential oil and rapeseed oil on fatty acid profile of thigh meat in broilers (%)

Items	C14:0	C14:1	C16:0	C16:1	C18:0	C18:1	C18:2	C18:3	C20:1	C20:2	C22:0	C22:6	U	S	U/S ratio
Main effects
RO levels
1%	0.48a	0.048	21.41a	3.74a	2.50	44.4b	24.04	0.141b	2.52b	0.148	0.33	0.148	75.05b	24.73a	3.05b
3%	0.47a	0.036	21.38a	3.55ab	2.60	44.7b	23.79	0.155b	2.61b	0.143	0.35	0.155	75.02b	24.81a	3.04b
5%	0.42b	0.033	19.28b	3.26b	2.48	48.5a	22.16	0.198a	3.02a	0.131	0.36	0.155	77.31a	22.56b	3.44a
SEM	0.006	0.004	0.34	0.106	0.10	0.96	0.89	0.01	0.068	0.005	0.024	0.016	0.39	0.39	0.068
TEO levels
0 ppm	0.48a	0.041	21.61a	3.82a	2.31b	43.06b	25.43a	0.146b	2.48b	0.146	0.32	0.123	75.13	24.73	3.07
300 ppm	0.45b	0.035	19.70b	3.3b	2.84a	49.62a	20.26b	0.186a	2.92a	0.138	0.34	0.168	76.47	23.35	3.28
500 ppm	0.44b	0.041	20.76ab	3.43b	2.43b	44.96b	24.30a	0.161ab	2.75a	0.138	0.38	0.166	75.79	24.02	3.18
SEM	0.006	0.004	0.34	0.106	0.10	0.96	0.89	0.01	0.068	0.005	0.024	0.016	0.39	0.39	0.068
RO levels × TEO levels
1%
0 ppm	0.490abc	0.050	22.01ab	3.89abc	2.26bc	40.72b	27.37ab	0.135b	2.39df	0.155	0.35b	0.135bcd	74.71bcd	25.11abc	2.97cde
300 ppm	0.440d	0.045	19.42cde	3.99ab	1.93e	46.93a	23.58abc	0.150b	2.94bcd	0.145	0.30b	0.115 cd	72.89d	26.98a	2.70e
500 ppm	0.510a	0.030	23.39a	3.58bcd	2.76ab	41.52b	25.35ab	0.155b	2.11f	0.140	0.32b	0.120 cd	77.79a	22.10de	3.53a
3%
0 ppm	0.495ab	0.055	23.38a	4.16a	1.98c	41.92b	25.18ab	0.135b	2.18f	0.130	0.27b	0.100d	76.66ab	22.97de	3.34ab
300 ppm	0.380e	0.025	17.74e	2.48e	3.22a	51.50a	20.10 cd	0.135b	3.46a	0.140	0.50a	0.240a	76.46ab	23.34cde	3.27abc
500 ppm	0.455 cd	0.045	21.17bc	3.64abcd	2.10c	41.47b	27.62a	0.290a	2.61de	0.145	0.38ab	0.16abcd	76.29ab	23.74cde	3.22abc
5%
0 ppm	0.455 cd	0.040	18.86de	3.18d	3.27a	50.58a	19.57 cd	0.155b	2.99bc	0.160	0.39ab	0.210ab	73.78 cd	26.12ab	2.83de
300 ppm	0.460bcd	0.030	20.67bc	3.30d	2.31bc	47.10a	22.80bcd	0.180b	2.66cde	0.110	0.29b	0.110 cd	75.72bc	24.11bcd	3.14bcd
500 ppm	0.450d	0.035	19.59 cd	3.42 cd	2.94a	51.19a	18.41d	0.150b	3.12ab	0.145	0.36b	0.185abc	77.86a	21.84e	3.56a
SEM	0.011	0.007	0.589	0.184	0.175	1.67	1.54	0.017	0.118	0.01	0.042	0.028	0.69	0.688	0.119
P value
RO	< 0.0001	0.06	0.01	0.01	0.69	0.01	0.29	0.002	0.0001	0.14	0.66	0.94	0.008	0.001	0.0008
TEO	0.004	0.47	0.04	0.007	0.004	0.004	0.001	0.04	0.0009	0.51	0.24	0.12	0.085	0.074	0.128
RO × TEO	0.0007	0.21	< 0.0001	0.003	< 0.0001	0.003	0.013	0.0004	< 0.0001	0.058	0.012	0.008	0.004	0.0025	0.004

SEM standard error of mean, TEO thymus essential oil, RO rapeseed oil, U total unsaturated, S total saturated

^a–cMeans with no common superscript within each column are significantly (P < 0.05) different

Sausage quality measurements

Sausage proximate composition

The results for the proximate composition of moisture, crude fat, crude ash, and crude protein are presented in Table 3. Meat sources, REO levels and interaction of meat sources and REO levels had no effect on proximate composition of sausage.

Table 3.

Effects of different sources of meat and levels of REO on proximate composition, color parameters and microbial total counts of chicken sausages

Items	Proximate composition (%)				Color parameters			Microbial total count
	Moisture	Crude fat	Crude ash	Crude protein	a*	b*	L*
Main effects
Meat sources
Meat of chicken fed with TEO	67.15	6.31	2.426	15.27	70.26	74.66a	42.66	37.60
Meat of chicken fed without TEO	67.15	6.32	2.426	15.32	70.12	73.63b	42.81	37.32
SEM	0.006	0.007	0.004	0.039	0.259	0.168	0.161	0.433
REO levels
Without REO or nitrite	67.14	6.31	2.426	15.23	23.03c	33.76c	48.17a	70.21a
120 ppm nitrite	67.16	6.32	2.431	15.33	95.07a	99.84a	38.47c	23.25c
120 ppm REO	67.14	6.30	2.426	15.35	68.10b	63.13b	45.95b	32.27b
60 ppm REO + 60 ppm nitrite	67.15	6.32	2.423	15.26	94.54a	99.84a	38.35c	24.10c
SEM	0.009	0.01	0.005	0.055	0.366	0.237	0.228	0.613
Meat sources × REO levels
Without REO or nitrite	67.14	6.30	2.425	15.23	23.03	34.39	48.02	70.80
Meat of chicken fed with TEO
120 ppm nitrite	67.16	6.31	2.425	15.27	95.29	100.05	38.42	22.90
120 ppm REO	67.14	6.30	2.427	15.30	68.10	64.14	45.88	32.35
60 ppm nitrite + 60 ppm REO	67.15	6.33	2.430	15.29	94.61	100.05	38.31	24.35
Without REO or nitrite	67.15	6.32	2.427	15.23	23.04	33.14	48.32	69.63
Meat of chicken fed without TEO
120 ppm nitrite	67.17	6.33	2.437	15.39	94.86	99.63	38.52	23.60
120 ppm REO	67.14	6.31	2.425	15.41	68.10	62.12	46.02	32.20
60 ppm nitrite + 60 ppm REO	67.15	6.30	2.417	15.23	94.48	99.63	38.40	23.85
SEM	0.013	0.015	0.008	0.078	0.518	0.336	0.322	0.867
P value
Meat sources	0.84	0.72	0.99	0.43	0.7	0.0002	0.50	0.65
REO levels	0.30	0.71	0.82	0.38	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Meat sources × REO levels	0.81	0.42	0.48	0.62	0.96	0.074	0.98	0.75

SEM standard error of mean, REO rosemary essential oil, TEO thymus essential oil

^a–cMeans with no common superscript within each column are significantly (P < 0.05) different

Due to the same formulation in the process of sausage production and also, the same primary source of sausage (chicken meat) and the same nutrition of chickens (except for antioxidant levels) in present study, the absence of significant changes was not unexpected. These results are consistent with the Viuda-Martos et al. (2010) which there was no significant change in moisture, ash, fat and protein by adding oregano essential oil to sausages.

Color measurement parameters of sausage

Based on the Table 3, sausages produced by meat from chicken fed TEO had highest (P = 0.0002) yellowness (b*) than meat from chicken fed without TEO. The redness (a*) and yellowness (b*) indexes were significantly higher (P < 0.0001) in sausages supplemented by REO:nitrite (60:60 ppm) and nitrite (120 ppm) followed by REO (120 ppm) than without REO and nitrite. Meat color is one of the most important parameters to evaluating meat and meat products quality. It is well known that oxidation of red pigments of meat products during the storage decrease redness index (Al-Marazzeq et al. 2015), while yellowness index is related to the existence of pigments but not to process of oxidation (El-Gharably and Ashoush 2012). Also, the preservation time did not effect on the yellowness index (Ashley 2013). The lightness (L*) index of sausage decreased by supplementation of REO:nitrite (60:60 ppm) and nitrite (120 ppm) than REO (120 ppm) and without REO and nitrite. The highest L* index of sausage were observed in without REO and nitrite sausages. Many factors such as water content, the type and concentration of pigments, and the absorption of moisture content of matrix-soluble substances are related to the lightness (L*) factor in sausage (El-Gharably and Ashoush 2012). Al-Marazzeq et al. (2015) claimed the lightness index increased over time, and nitrite-applied treatments with olive leaves extract had more lightness than the control group. In opposite, Khaleghi et al. (2016) reported that with the replacement of nitrite in sausages by black barberry, lightness decreased comparing with the control group. Increasing of index a* during the storage period because of the addition of rosemary and Chinese mahogany to chicken sausages was reported by Liu et al. (2009). El-Gharably and Ashoush (2012) found that index a* during storage decreased by addition of pomegranate rind and red beet powders to veal sausage. Al-Marazzeq et al. (2015) showed that the least yellowness index during the period of storage was pertinent with the control sample followed by samples containing 60 and 80 ppm nitrite or extract of olive leaves.

Microbiological analysis of sausage

As shown in Table 3, chicken meat sources (containing TEO or without TEO) and interaction of meat and antioxidant (nitrite or REO) had no effect on microbial count, while addition of antioxidant during sausage production changed microbial load (P < 0.0001). The lowest microbial total counts of sausage were observed by nitrite (120 ppm) and REO:nitrite (60:60 PPM) < REO (120 ppm) < without REO and nitrite (P < 0.0001). In line with Riazi et al. (2016) demonstrated that as increasing in nitrite levels, the microbial load of the sausages samples decreased as comparison to control sample, followed by nitrite (60 ppm + 1 to 2% red grape pomace). In opposite, Khaleghi et al. (2016) found that the sausages containing the black barberry extract (90 ppm) showed a lower overall microbial count than control group containing 60 ppm nitrite during the storage. Viuda-Martos et al. (2010) reported that dietary fiber + oregano essential oil reduced aerobic and lactic acid bacteria counts of sausages stored for 24 days. Sojic et al. (2018) reported that the addition of nutmeg essential oil in amounts of 10 and 20 ppm can be successfully applied to decreasing total number of aerobic mesophilic bacteria in cooked sausages after 60 days of storage (137 and 78.3 cfu/g vs. 185 cfu/g in control).

Sausage lipid oxidation

Based on the Table 4, the MDA concentrations of sausages were not affected by interaction of meat source and REO supplementation. The using of meat from chicken fed with TEO reduced the MDA content in the sausages as compared to meat from chicken fed with TEO sample on the 7th day of storage (P < 0.05).The MDA concentrations of REO and nitrite free sausages were significantly higher (P < 0.0001) than the sausages supplemented with REO and nitrite at days 0, 7, 14, 21, 28 and 35. The sausages supplemented with REO (120 ppm) had the lowest (P < 0.001) MDA concentration, followed by nitrite: REO (60:60 ppm) < nitrite (120 ppm). MDA concentrations were lower (P < 0.001) in sausages containing REO (60 ppm) and nitrite (60 ppm) compared with sausages supplemented with nitrite (120 ppm). It is well documented that antioxidant activity of spice, thyme and rosemary oils may interfere with the enzymatic systems involved in initiation reactions (You et al. 1999) as well as prevent free radicals in many products (Viuda-Martos et al. 2010). Base on the Table 4, all MDA values were below 1 mg MDA/kg reported by Ockerman (1976) as emergence of rancidness in meat products. Moreover, replacement of nitrite in sausages by black barberry decreased the TBA index from the days of 9–23 (Khaleghi et al. 2016). Sojic et al. (2018) found that lipid oxidative changes at the beginning of the storage (1st day) were within a very narrow range between products containing or free of antioxidant, while during storage, TBARS values increased in all products with very gentle slope for which supplemented with antioxidant. In this case, Yanishlleva et al. (2006) reported green tea and thyme extracts reduced TBARS values of luncheon meat samples compared to control sample. It is well demonstrated that phenolic mono-terpenes in thyme, thymol and carvacrol inhibit lipid oxidation.

Table 4.

Effects of different sources of meat and levels of REO on lipid oxidation of chicken sausages [all data points are mean malondialdehyde (MDA) concentrations (mg MDA/kg chicken sausages)]

Items	0 day	7 days	14 days	21 days	28 days	35 days
Main effects
Meat sources
Meat of chicken fed with TEO	0.097	0.213b	0.485	0.338	0.260	0.196
Meat of chicken fed without TEO	0.103	0.217a	0.486	0.341	0.262	0.198
SEM	0.002	0.001	0.001	0.001	0.001	0.0009
REO levels
Without REO or nitrite	0.163a	0.278a	0.645a	0.505a	0.398a	0.328a
120 ppm nitrite	0.100b	0.228b	0.498b	0.331b	0.249b	0.189b
120 ppm REO	0.060d	0.163d	0.389d	0.232d	0.184d	0.111d
60 ppm REO + 60 ppm nitrite	0.078c	0.192c	0.409c	0.290c	0.211c	0.160c
SEM	0.003	0.001	0.002	0.001	0.001	0.001
Meat sources × REO levels
Meat of chicken fed with TEO
Without REO or nitrite	0.159	0.276	0.644	0.503	0.397	0.327
120 ppm nitrite	0.096	0.226	0.498	0.330	0.249	0.188
120 ppm REO	0.057	0.161	0.388	0.231	0.183	0.110
60 ppm nitrite + 60 ppm REO	0.075	0.191	0.408	0.289	0.211	0.159
Meat of chicken fed without TEO
Without REO or nitrite	0.166	0.281	0.645	0. 507	0.399	0.329
120 ppm nitrite	0.103	0.230	0.499	0.332	0.250	0.190
120 ppm REO	0.063	0.165	0.390	0.232	0.184	0.112
60 ppm nitrite + 60 ppm REO	0.081	0.194	0.411	0.292	0.212	0.161
SEM	0.004	0.002	0.003	0.002	0.002	0.001
P value
Meat sources	0.06	0.01	0.51	0.23	0.26	0.2
REO levels	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Meat sources × REO levels	0.99	0.95	0.99	0.97	0.99	0.98

SEM standard error of mean, REO rosemary essential oil, TEO thymus essential oil

^a–cMeans with no common superscript within each column are significantly (P < 0.05) different

Sausage pH measurement

The effects of different sources of meat and antioxidant on pH level of chicken sausages are shown in Table 5. The meat of chicken fed TEO had higher (P < 0.05) values of pH than meat of chicken fed without TEO samples. Supplementation of antioxidant increased pH index as compared to without REO and nitrite group, such that the highest (P < 0.0001) pH value was related to REO (120 ppm), followed by REO:nitrite (60:60) and nitrite (120 ppm). Supplementation of essential oils as nitrite substitute could be an acceptable alternative for reducing the intake of nitrite in processed meats to alleviate the potential risk of the formation of any N-nitroso compounds (Karolyi 2003). Interaction of meat from chicken do not fed TEO and REO:nitrite (60:60) increased pH values on days 28 and 35. The highest reduction of pH index was observed on day 21 of storage, which was probably related to the production of organic acids by bacteria, together with the oxidation of fat and the development of acidic compounds (Liu et al. 2009).

Table 5.

Effects of different sources of meat and levels of REO on pH of chicken sausages

Items	0 day	7 days	14 days	21 days	28 days	35 days
Main effects
Meat sources
Meat of chicken fed with TEO	6.21a	6.18a	6.09a	5.91	5.99a	6.16a
Meat of chicken fed without TEO	6.20b	6.17b	6.08b	5.92	5.98b	6.10b
SEM	0.002	0.002	0.002	0.002	0.022	0.001
REO levels
Without REO or nitrite	6.16c	6.14c	6.05c	5.82b	5.93c	5.99b
120 ppm nitrite	6.20b	6.19b	6.09b	5.95a	5.98b	6.00b
120 ppm REO	6.23a	6.20a	6.11a	5.95a	6.01a	6.02a
60 ppm REO + 60 ppm nitrite	6.22b	6.187ab	6.09b	5.94a	6.02a	6.03a
SEM	0.002	0.004	0.004	0.003	0.002	0.002
Meat sources × REO levels
Meat of chicken fed with TEO
Without REO or nitrite	6.16	6.14	6.05	5.83	5.93e	6.00d
120 ppm nitrite	6.20	6.18	6.09	5.95	5.98d	6.00d
120 ppm REO	6.23	6.19	6.10	5.95	6.01b	6.02b
60 ppm nitrite + 60 ppm REO	6.21	6.17	6.08	5.95	6.00c	6.01c
Meat of chicken fed without TEO
Without REO or nitrite	6.17	6.15	6.06	5. 82	5.93e	5.99d
120 ppm nitrite	6.21	6.18	6.09	5.95	5.98d	6.00d
120 ppm REO	6.24	6.21	6.12	5.95	6.02b	6.02b
60 ppm nitrite + 60 ppm REO	6.22	6.20	6.11	5.94	6.04a	6.04a
SEM	0.004	0.005	0.005	0.004	0.004	0.003
P value
Meat sources	0.002	0.001	0.002	0.69	0.002	0.02
REO levels	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Meat sources × REO levels	0.62	0.31	0.3	0.98	< 0.0001	< 0.0001

SEM standard error of mean, REO rosemary essential oil, TEO thymus essential oil

^a–cMeans with no common superscript within each column are significantly (P < 0.05) different

Sausage sensory evaluation

In assessing different sources of meat and antioxidants, the sensory properties of the samples were determined by the panelists, which were not significantly different in terms of color, taste, smell and texture at 5% confidence level (P > 0.05) (Fig. 1). This could be due to the various compounds used in sausage formulation and the overlapping effect of REO and nitrite with the other compounds. El-Gharably and Ashoush (2012) reported that the sensory features (other than taste and water content) were not affected by the type and amount of compounds of pomegranate rind powder and red beet powder. Jin et al. (2014) reported that beet powder addition, other than color, did not significantly effect on sensory features of sausages. No changes in sensory features of sausages by replacing of nitrite with black barberry in sausage samples reported by Khaleghi et al. (2016). Sojic et al. (2018) reported that during 60 days of storage, sensory quality of odor decreased in sausage samples, while sensory evaluated odor remained higher for 20 ppm than 0 and 10 ppm nutmeg essential oil, which may be correlated with minor lipid oxidation in sausages.

Fig. 1.

Sensory evaluation analysis of sausage substituted nitrite with REO, REO: rosemary essential oil, Control: meat of chicken fed without TEO, Rich meat: meat of chicken fed with TEO

Conclusion

The results of the present study indicated that fatty acid profile of meat could be altered by adding rapeseed oil to the feed of broilers and thymus essential oil can extend the shelf life and improve the quality of meat and meat products. Moreover, feeding of poultry with a higher level of natural dietary antioxidants provides the poultry industry with a simple method for improvement of the oxidative stability, shelf life and acceptability of poultry meats such as sausage. Furthermore, according to the obtained results and also considering the health characteristics of the natural ingredients used, as well as the losses caused by synthetic preservatives for human health, it is possible to replace a part of nitrite (at least 60 ppm) with rosemary essential oil together with the desirable preservation of all the physical, chemical, microbial and sensory properties, which could be an effective step to improve the quality and health of meat products. Finally, the use of meat of chickens fed with thymus essential oil improved most of the traits of sausages such as lipid oxidation and pH value.

Compliance with ethical standards

Conflict of interest

No potential conflict of interest was reported by the authors.

Ethics statement

We declare no ethical issue related with this article.