Producing an Emulsified Meat System by Partially Substituting Pig Fat with Nanoemulsions that Contain Antioxidant Compounds: The Effect on Oxidative Stability, Nutritional Contribution, and Texture Profile.

The objective of this study was the incorporation of a water-oil (W/O) nanoemulsion for the partial substitution of pig fats and the addition of antioxidant compounds in an emulsified meat system (EMS). The nanoemulsion was formulated with orange essential oil and cactus acid fruit (xoconostle). The treatments were different percentages (0, 1, 2, 3, 4, and 5%) of the nanoemulsion for the substitution of pig fat in the EMS. The proximal analysis (moisture, protein, fat, and ash), texture profile (hardness, cohesiveness, springiness, and chewiness), phenolic compounds and antioxidant capacity 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and 2-thiobarbituric acid reactive substances (TBARS) were evaluated. All variables showed significant differences (p < 0.05). The results for protein, fat, and ash exhibited increments with the addition of the nanoemulsion, and moisture loss was reduced. The profile showed increments in hardness and chewiness. The addition of the nanoemulsion incremented the phenolic compounds and antioxidant capacity (DPPH and ABTS), decreased production of Malonaldehyde, and reduced lipid oxidation. The result of the addition of the nanoemulsion in the EMS is a product with a substantial nutritional contribution, antioxidant capacity, and excellent shelf life.

1. Introduction

In the meat industry, fat is very important for emulsified products. Fat is responsible for emulsion stability and water retention capacity, further providing energy, essential fatty acids, and fat soluble vitamins [1]. The fat soon degrades due to oxidation, thereby producing poor sensory characteristics, discoloration, and rancidity [2]; in addition, fat oxidation results in a reduction in shelf life and production of toxic compounds [3].

Emulsified meat products are enhanced with synthetic antioxidant compounds, including butyl-hydroxytoluene, butyl-hydroxyanisole, and t-butyl-hydroxyquinone, among others, to reduce fat oxidation and extend shelf life [4]. These synthetic products have the disadvantages of promoting toxicological, mutagenic, and carcinogenic effects [5,6,7]. One alternative option is the use of natural antioxidants in meat products [2].

The cactus acid fruit, xoconostle, from the genus Opuntia, contains phenolic compounds, carotenoids, betacyanins, and betalains [8]. These bioactive compounds have shown antioxidant activity [9] and antibacterial activity [10]. Furthermore, the orange essential oils include terpenes, such as D-limonene, that protect fat against oxidizing compounds [11]. In addition, essential oils have been shown to possess antibacterial, antifungal, and antioxidant activities [12,13], so these components can be used as functional ingredients in foods [14,15]. The antioxidant compounds are sensitive to external factors, such as light, temperature, and oxygen. One way to protect these compounds is by using encapsulation, such as in the form of nanoemulsions. This type of encapsulation has the advantage of improving the transportation and controlling the release of active molecules through the biological membrane [16].

Sharma et al. [17] incorporated four types of essential oils (clove, holy basil, cassia, and thyme) in emulsified chicken and demonstrated a reduction in fat oxidation. Wang et al. [3] substituted pig fat with camellia oil gel in sausage and found favorable results, such as reduced fat, lower moisture, and minor values of 2-thiobarbituric acid reactive substances (TBARS).

The objective of this work was the partial substitution of pig fat with water-oil W/O emulsions containing cactus acid fruit (xoconostle) in an emulsified meat system to evaluate the physicochemical characteristics, texture profile, and oxidative stability for 60 days. The objective of this work was to evaluate the effect of the partial substitution of pig fat with W/O emulsions containing cactus acid fruit (xoconostle) in an emulsified meat system on physicochemical characteristics, texture profile, and oxidative stability.

2. Materials and Methods

2.1. Preparation of the Nanoemulsion

The nanoemulsion was water in oil (W/O). It was prepared according to the methodology of Guler et al. [18] with some modifications. The continuous phase was orange essential oil (Hilmar Ingredients, USA) (70%), the dispersed phase was the cactus acid fruit (xoconostle) (10%), and the surfactant was liquid soya lecithin (Hilmar Ingredients, USA) (20%). All components were stirred using an ultrasonic processor (Ultrasonic Sonics Vibra-Cell, VCX 130, USA). A 6 mm probe was used and 20 intervals (20 s/interval) of sonication and recesses of 10 s were established to obtain the necessary drop size. The ultrasonic processor was used at 80% amplitude at a frequency of 20 kHz, and the mixture was placed in an ice bath to avoid temperature increases during mixing. The droplet size distribution of the nanoemulsion was determined with the dynamic laser light scattering technique using Zetasizer equipment (Nano-ZS2000 Model Malvern Instruments Ltd. Malvern, Worcestershire, United Kingdom) by placing the sample in a glass cell. Five replicates were considered for each formulation [19]. Furthermore, the phenolic content and antioxidant activity (2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) were determined in the nanoemulsions.

2.2. Production of the Emulsified Meat System

The emulsified meat system was carried out according to the method described by Cofrades et al. [20] with some modifications. Formulations with different percentages of animal fat and nanoemulsion were made (Table 1). The minced meat (1 cm2), salt, and ice were placed in a cutter (Dito-Sama F 23200 GBR, Aubusson, France) and beaten for two minutes, and then nanoemulsion was added to the mixture and beaten for one minute more.

Table 1

Formulation of emulsified meat systems with different percentages of nanoemulsion.

Treatments	Meat %	Fat %	Nanoemulsion %	Ice %	Salt %
0%	65	20	0	13	2
EMSN 0%	65	19	1	13	2
EMSN 2%	65	18	2	13	2
EMSN 3%	65	17	3	13	2
EMSN 4%	65	16	4	13	2
EMSN 5%	65	15	5	13	2

Emulsified meat system with nanoemulsion (EMSN).

The fat was incorporated into the mixture, maintaining a temperature no higher than 16 °C. The mixture was fed through a stuffing device (BG-PRUFRZERT Inc., City of México, México) and injected into 20 mm diameter synthetic cellulose casings (Viscofan Brand Inc., City of México, México). The filled casings were heated to 72 °C for 30 min, and then subjected to thermal shock by being placed in ice. Finally, the meat emulsion in casings were vacuum packed in bags (Zubex Inc., City of México, México) in a sealer (Tor Rey EVD48, City of México, México) and refrigerated at 4 °C.

2.3. Proximal Composition

The proximal analysis was performed according to the official methods of the Association of Official Agricultural Chemists (AOAC) edited by Horwitz [21]. The moisture was calculated by drying a sample in a stove at 100 °C for 8 h (Official Method 925.09), the fat content by the Soxhlet method (Official Method 923.05), the ash percentage was determined by the incineration of the muffle samples at 550° C for 8 h (Official Method 923.03), and the protein content by the Kjeldahl method (Official Method 981.10).

2.4. Texture Profile Analysis (TPA)

These tests were performed according by Cofrades et al. [22] with some modifications. Eight repetitions were performed for each treatment. Cubes of 1 × 1 × 1 centimeters were elaborated and a texturometer (Brookfield CT3 texture analyzer, Brookfield Engineering Laboratories, Inc. Middleboro, MA, USA) was used. The samples were axially compressed to 50% of their original height with a 4.5 kg load cell at a speed of 1 mm/s, with the use of a TA3/1000 probe and a TA-BT-KI table. The parameters measured were hardness, cohesiveness, springiness, and chewiness. The test was performed at room temperature.

2.5. Total Phenols

The content of total phenols was done following a modified version of the methodology by Singleton et al. [23]. The samples were diluted to 1:10. Then, 0.5 mL of sample was mixed with 2.5 mL of previously diluted (1:10) Folin-Ciocalteau reagent (Sigma-Aldrich, St. Louis, MO, USA) and 2 mL of 7.5% sodium carbonate (Fermont) was added. The mixture was left for 120 min in total darkness. After, the samples were read in a spectrophotometer (JENWAY 6715 Ultraviolet/Visible (UV/V), Staffordshire, UK) at a wavelength of 760 nm. The results were expressed as mg of gallic acid equivalents for 100 g of emulsified meat system with nanoemulsion (EMSN) (GAE/100 g of EMSN).

2.6. DPPH

The methodology of Brand-Williams et al. [24] for DPPH test was used with some modifications. Here, 0.0039 g of DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, USA) in 50 mL of 80% methanol (JT Baker, VWR International. Tultitlán, México) was mixed and left for 2 h in the dark, then calibrated at 0.7 ± 0.1 absorbance. Then, 0.5 mL of this mixture was added to 2.5 mL of DPPH solution and left in darkness for 60 min. After, the samples were read at 517 nm in a spectrophotometer (JENWAY 6715 UV/V, UK). The results were expressed as mg of ascorbic acid equivalents for 100 g of emulsified meat system with nanoemulsion (EMSN) (AAE/100 g of EMSN).

2.7. ABTS

Here, 7 mM (10 mL) of 2, 2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-Aldrich, St. Louis, MO, USA) and 2.45 mM (10 mL) of potassium persulfate were mixed. The mixture was left in complete darkness for 16 h. Next, the mixture was adjusted with 20% ethanol to obtain a value of 0.7 ± 0.1 absorbance. The final solution (3.9 mL) was taken and 100 μL of sample was added. The mixture was read at 734 nm [25]. The results were expressed as mg of ascorbic acid equivalents for 100 g of emulsified meat system with nanoemulsion (EMSN) (AAE/100 g of EMSN).

2.8. 2-thiobarbituric acid reactive substances (TBAR)

Lipid oxidation was evaluated according to Wang et al. [26] with some modifications. An extractor solution was prepared containing 7.5 % trichloroacetic acid (Fermont PA Cert, Monterrey, México), 0.1% gallic acid (Fermont PA Cert, Monterrey, México), and 0.1 % EDTA, disodium salt dehydrate (Baker ACS, México). Then, 2.5 g of samples were taken and homogenized with 25 mL of extractor solution in an Ultraturrax T25 (IKA-Werke GmbH & Co. KG) 3000 rpm for 1 min. The homogenate was centrifuged at 6000× g forces at 20 °C for 10 min. The supernatant (2 mL) was mixed with 80 mM (2 mL) of thiobarbituric acid (BP 50067 lllkirch, Strasbourg, France) (TBA). The mixture was incubated at 40 ° C for 90 min and it was read at 532 nm. The TBARS values were interpreted with the calibration curve of 1,1,3,3-tetramethoxypropane (Malonaldehyde) (Sigma-Aldrich, St. Louis, MO, USA) in different concentrations and the results were expressed in milligrams of malonaldehyde (MDA)per kilogram of sample (mg MDA /Kg).

2.9. Statistical Analysis

The experimental design was completely random. The results were analyzed by ANOVA, when there were significant differences (p < 0.05), comparison of media (Tukey) was used with the statistical program STATGRAPHICS C. XVI Version 16.1.03 (Statgraphics Technologies Inc., The Plains, VA, USA).

3. Results and Discussion

3.1. Nanoemulsions and Characterization

The drop diameter was 73 ± 6 nm and the Z potential value was −107 mV. Both parameters are characteristic of nanoemulsions [27,28]. Our results are similar to those reported in Gago et al. [29] for nanoemulsions of clove and lemongrass essential oil. The phenolic content was 184.3 mg GAE/100 g, the antioxidant activity from DPPH was 97.76 mg AAE/100 g, and ABTS was 126.3 mg AAE/100g in the nanoemulsions.

3.2. Proximate Composition

Significant differences (p < 0.05) were observed in the moisture of the meat emulsion system in the different treatments and times. Treatments with the nanoemulsions demonstrated a reduced loss of moisture (Table 2), and similar results were reported by Sharma et al. [17] in chicken sausage with the addition of different essential oils. The major reason for the retention of water could be that the nanoemulsions contain soy lecithin in the formulation, which was used as an emulsifier [30,31].

Table 2

Proximal composition of the emulsified meat system with nanoemulsion for the parameters of moisture, protein, fat and ash.

	Days	EMSN 0%	EMSN 1%	EMSN 2%	EMSN 3%	EMSN 4%	EMSN 5%
Moisture	1	68.58 ± 0.085 aC	68.53 ± 0.007 aB	68.52 ± 0.019 aD	68.59 ± 0.094 aB	68.65 ± 0.093 aE	68.67 ± 0.092 aD
	15	67.88 ± 0.018 aC	67.94 ± 0.479 aB	67.98 ± 0.099 aC	67.92 ± 0.382 aB	67.98 ± 0.013 aD	67.96 ± 0.112 aC
	30	65.14 ± 0.427 aB	65.60 ± 0.107 aA	65.72 ± 0.077 aB	65.89 ± 0.036 aA	66.05 ± 0.022 aC	66.11 ± 0.083 aB
	45	64.46±0.252 aAB	65.10 ± 0.075 bA	65.23 ± 0.001 bA	65.32 ± 0.001 bcA	65.67± 0.009 cdB	65.80 ± 0.003 dA
	60	63.58 ± 0.002 aA	64.95 ± 0.002 bA	65.13 ± 0.010 cA	65.24 ± 0.011 dA	65.40 ± 0.017 eA	65.58 ± 0.009 fA
Protein	1	14.89 ± 0.020 aA	15.09 ± 0.008 abA	15.10± 0.024 abA	15.15±0.009 abcA	15.29± 0.146 bcA	15.40 ± 0.103 cA
	15	15.39±0.041 aB	15.47±0.199 aAB	15.53 ± 0.073 aB	15.63 ± 0.024 aB	15.67 ± 0.306 aA	15.83 ± 0.056 aB
	30	15.49± 0.022 aBC	15.54 ± 0.031 aB	15.61 ± 0.089 aB	15.84 ± 0.017 bC	15.87± 0.037 bAB	16.00 ± 0.035 bB
	45	15.55 ± 0.005 aC	16.08 ± 0.066 bC	16.24 ± 0.023 cC	16.36 ± 0.024 cdD	16.41± 0.054 dBC	16.46 ± 0.035 dC
	60	16.15 ± 0.059 aD	16.65 ± 0.003 bD	16.69 ± 0.002 bD	16.84 ± 0.008 cE	16.89 ± 0.008 cC	16.94 ± 0.012 cD
Fat	1	8.83 ± 0.058 aA	9.39 ± 0.007 bA	10.26 ± 0.056 cA	10.60 ± 0.077 dA	11.74 ± 0.012 eA	12.23 ± 0.092 fA
	15	9.35 ± 0.186 aB	10.11 ± 0.037 bA	10.87 ± 0.051 cA	11.12 ± 0.070 cB	12.44 ± 0.147 aB	12.61 ± 0.192 dA
	30	13.38 ± 0.024 aC	14.29 ± 0.257 abB	14.43 ± 0.370 bB	14.75 ± 0.026 bC	14.90 ± 0.322 bC	15.32 ± 0.325 bB
	45	13.95 ± 0.061 aD	14.53 ± 0.646 abB	14.59 ±0.087 abB	14.81± 0.008 abC	15.21 ± 0.099 bC	15.29 ± 0.015 bB
	60	14.52 ± 0.008 aE	14.64 ± 0.068 abB	14.78 ± 0.022 bB	14.95 ± 0.011 dA	15.33 ± 0.012 dC	15.69 ± 0.024 eB
Ash	1	1.94 ± 0.037 aA	1.94 ± 0.017 aA	1.94 ± 0.001 aA	1.94 ± 0.005 aA	1.95 ± 0.001 aA	1.95 ± 0.006 aA
	15	1.95 ± 0.002 aA	1.96 ± 0.001 abAB	1.96 ± 0.002 abcA	1.97 ± 0.003 bcB	1.97 ± 0.002 cB	1.97 ± 0.002 cB
	30	1.96 ± 0.001 aA	1.96 ± 0.002 aAB	1.96 ± 0.007 aB	1.97 ± 0.001 abB	1.98 ± 0.001 abB	1.98 ± 0.003 bB
	45	1.97 ± 0.007 aA	1.97 ± 0.007 aAB	1.97 ± 0.002 aBC	1.97 ± 0.001 aB	1.98 ± 0.001 aB	1.98 ± 0.003 aB
	60	1.98 ± 0.002 aA	1.98 ± 0.006 aB	1.98 ± 0.001 aC	1.98 ± 0.004 aB	1.98 ± 0.006 aB	1.98 ± 0.003 aB

Emulsified meat system with nanoemulsion (EMSN). The lowercase letters in the superscript indicate significant differences (p < 0.05) between treatments (rows), and uppercase letters indicate significant differences in each treatment with respect to time (columns) (p < 0.05).

Significant differences (p < 0.05) were observed in protein between the different treatments and times. The major protein content was observed in the treatments with nanoemulsions (Table 2). Choi et al. [1] found similar results in the substitution of pig fat with vegetable oil in the emulsified meat system. However, Bolger, Brunton, and Monahan [32] did not find significant differences (p > 0.05) in protein content in an emulsified product with encapsulated flaxseed oil. The increment in protein could be due to soy lecithin, which contains amino acids.

The EMSN showed a significant increment (p < 0.05) in the content of fat after the addition of the nanoemulsion (Table 2). In contrast, Choi et al. [1] found less fat with the addition of vegetable oils in EMS; however, the quality of the lipid provided by the nanoemulsion is better compared to that provided by pig fat. The orange essential oil contains antioxidant compounds, such as D-limonene, according to Chasquibol et al. [11].

The values of ash were between 1.94 and 1.95 in the treatment with EMSN 5% (Table 2). Choi et al. [1] reported similar results (1.72 to 1.97) with the addition of vegetables oils in EMS.

3.3. Texture Profile Analysis (TPA)

The nanoemulsion significantly (p < 0.05) affected the hardness of the EMSN. Treatment with the 5% nanoemulsion produced the most substantial hardness (Table 3). Similar results were reported by Youssef and Barbut [33] in a meat batter with canola oil. These authors attributed the increase in the hardness to the oil’s smaller globule size and the enhanced interaction between proteins.

Table 3

Texture profile analysis (TPA) for the parameters hardness, cohesiveness, springiness, and chewiness in the emulsified meat system with nanoemulsion.

	Days	EMSN 0%	EMSN 1%	EMSN 2%	EMSN 3%	EMSN 4%	EMSN 5%
Hardness (N)	1	12.49 ± 0.344 bA	12.38 ± 0.307 bA	12.63 ± 0.302 aA	13.44± 0.358 cA	14.11 ± 0.306 dA	14.57± 0.333 dA
	15	13.68 ± 0.238 bB	12.93 ± 0.357 aB	12.94 ± 0.406 aB	14.52 ± 0.270 cB	15.16 ± 0.254 dB	15.53 ± 0.252 dB
	30	14.40 ± 0.238 bC	13.51 ± 0.336 aC	14.13 ± 0.374 aB	15.43 ± 0.245 cC	16.57 ± 0.332 dC	16.47 ± 0.406 dC
	45	15.12 ± 0.681 cD	13.73 ± 0.165 aC	14.94 ± 0.373 bC	16.43 ± 0.261 dD	17.58 ± 0.313 eD	17.50 ± 0.288 eD
	60	17.76 ± 0.252 cE	14.59 ± 0.318 aD	15.25 ± 0.356 bD	17.58 ± 0.314 cE	18.40 ± 0.264 dE	18.52 ± 0.299 dE
Cohesiveness	1	0.65 ± 0.007 abC	0.65 ± 0.005 abC	0.64 ± 0.005 aD	0.65 ± 0.006 abC	0.64 ± 0.004 abD	0.65 ± 0.005 bC
	15	0.64 ± 0.004 bC	0.63 ± 0.011 abB	0.63 ± 0.007 aC	0.63 ± 0.007 abB	0.63 ± 0.007 abC	0.63 ± 0.006 abB
	30	0.63 ± 0.005 abB	0.63 ± 0.005 bB	0.62 ± 0.009 aBC	0.63 ± 0.004 abB	0.63 ± 0.006 abBC	0.63 ± 0.007 bB
	45	0.62 ± 0.007 abB	0.61 ± 0.014 abA	0.61 ± 0.010 aAB	0.62 ± 0.007 abA	0.62 ± 0.004 bB	0.62 ± 0.005 abA
	60	0.60 ± 0.011 aA	0.61 ± 0.010 abA	0.61 ± 0.009 abA	0.61 ± 0.007 abA	0.61 ± 0.008 abA	0.62 ± 0.006 bA
Springiness (mm)	1	4.36 ± 0.029 bA	4.33 ± 0.020 abC	4.34 ± 0.021 abC	4.32 ± 0.031 abD	4.31 ± 0.024 aB	4.31 ± 0.039 aB
	15	4.35 ± 0.020 dB	4.26 ± 0.014 aB	4.33 ± 0.021 cdC	4.30 ± 0.024 bcCD	4.29 ± 0.024 bAB	4.29 ± 0.015 bAB
	30	4.34 ± 0.018 cB	4.24 ± 0.017 aB	4.32 ± 0.019 cBC	4.27 ± 0.027 abBC	4.28 ± 0.023 abAB	4.28 ± 0.025 bAB
	45	4.26 ± 0.023 abCA	4.23 ± 0.033 aB	4.30 ± 0.019 cAB	4.24 ± 0.036 abAB	4.26 ± 0.029 abcA	4.27 ± 0.031 bcAB
	60	4.24 ± 0.024 bcA	4.18 ± 0.031 aA	4.28 ± 0.028 cA	4.21 ± 0.053 abA	4.25 ± 0.040 bcA	4.26 ± 0.030 cA
Chewiness (NXmm)	1	33.41 ± 1.25 aA	34.15 ± 0.887 bA	36.95 ± 0.543 bA	37.95± 0.358 cA	38.43 ± 0.990 cA	38.55± 0.525 cA
	15	34.64 ± 1.04 aA	35.24 ± 0.792 aB	37.96 ± 0.921 bA	38.97 ±0.542 bcA	39.29 ±0.831 cAB	41.01 ± 0.752 dB
	30	37.29 ± 1.04 bB	35.88 ±0.984 abC	42.00 ± 0.664 dB	41.64 ± 0.877 dB	40.14 ± 0.981 cB	42.72 ± 0.771 dC
	45	38.78 ± 0.84 bC	36.23 ± 0.900 aC	43.33± 0.928 cC	42.63 ± 0.850 cB	42.05 ± 0.870 cC	43.29 ± 0.880 cC
	60	41.26 ± 0.775 bD	37.23 ± 0.839 aD	45.98 ± 0.988 cD	45.31 ± 0.652 cC	45.15 ± 0.837 cD	46.25 ± 0.904 cD

Emulsified meat system with nanoemulsion (EMSN). The lowercase letters in the superscript indicate significant differences (p < 0.05) between treatments (rows). and uppercase letters indicate significant differences in each treatment with respect to time (columns) (p < 0.05).

The nanoemulsion did not affect the cohesiveness of the EMSN (Table 3). Wang et al. [3] reported the same results after the partial substitution of pig fat with camellia oil gel in sausage. In contrast, Choi et al. [1] observed an increment after the addition of vegetable oils in an EMSN.

No significant differences (p > 0.05) were observed between treatments with respect to springiness (Table 3). The incorporation of flaxseed oil did not affect the springiness of chicken sausage [32]. The EMSN did not show changes in springiness due to the addition of oils or nanoemulsions.

The EMSN exhibited a significant increment (p < 0.05) in chewiness after the incorporation of the nanoemulsion (Table 3). These results coincide with those reported by Youssef and Barbut [33], Choi et al. [1], and Bolger et al. [32] with respect to the substitution of fat with vegetable and seed oils in EMSN. The increase in chewiness is related to the protein incorporated within the nanoemulsion.

The effect on shelf life exhibited significant differences (p < 0.05) after the substitution of pig fat with the nanoemulsions. The EMSN showed increased hardness and chewiness but reduced cohesiveness and springiness, and these effects could be attributed to the loss of moisture during storage.

3.4. Total Phenols and Antioxidant Activity

The contents of phenols were significantly enhanced (p < 0.05) by the incorporation of the nanoemulsions (Table 4) because the nanoemulsions contain phenolic compounds from the xoconostle extract. The addition of cherry extract to sausage also increased the content of phenols [34].

Table 4

Phenols, antioxidant activity via the DPPH and ABTS methods, and oxidative stability via the TBARS method in an emulsified meat system with nanoemulsion.

	Days	EMSN 0%	EMSN 1%	EMSN 2%	EMSN 3%	EMSN 4%	EMSN 5%
Phenols mg GAE/100g	1	ND	12.76 ± 0.345 aC	13.29 ± 0.486 aC	15.64 ± 0.177 bD	19.86 ± 0.215 cD	24.93 ± 0.170 dE
	15	ND	12.22 ± 0.385 aC	14.39 ± 0.049 bD	14.47 ± 0.098 bC	15.21 ± 0.098 cC	18.09 ± 0.161 dD
	30	ND	11.25 ± 0.098 aB	12.31 ± 0.078 bB	12.39 ± 0.345 bB	15.10± 0.085 cC	16.04 ± 0.085 dC
	45	ND	10.45 ± 0.274 aA	11.71± 0.148 aAB	12.50 ± 0.098 bB	13.25 ± 0.090 cB	14.58 ± 0.085 dB
	60	ND	10.40 ± 0.098 aA	11.39 ± 0.098 bA	11.56 ± 0.177 bA	11.76 ± 0.085 bcA	12.16 ± 0.177 cA
DPPH mg AAE/100g	1	15.55 ±0.288 aC	18.13 ± 0.377 bD	19.20 ± 0.108 cD	19.26 ± 0.188 cD	19.76 ± 0.288 cD	19.89 ± 0.288 cD
	15	13.60 ± 0.188 aC	17.88 ± 0.474 bD	19.01 ± 0.474 cD	18.69 ± 0.188 cD	19.07±0.499 cdCD	19.89 ± 0.288 dD
	30	11.40 ± 0.474 aB	16.05 ± 0.201 bC	17.18 ± 0.343 cC	17.94 ± 0.218 dC	18.50 ± 0.288 deC	18.94 ± 0.288 eC
	45	11.77 ± 0.288 aB	11.84 ± 0.108 abB	12.34 ± 0.108 bB	14.92 ± 0.188 cB	15.30 ± 0.499 cB	16.24 ± 0.188 dB
	60	7.94 ± 0.188 aA	10.14 ± 0.288 bA	10.89± 0.288 bcA	11.52 ± 0.499 cA	12.53 ± 0.390 dA	14.48 ± 0.288 eA
ABTS mg AAE/100g	1	22.53 ± 0.492 aD	28.98 ± 0.372 bC	33.07 ± 0.492 cD	33.93 ± 0.322 cC	34.25± 0.445 cB	37.59± 0.492 dC
	15	20.92 ± 0.492 aC	26.73 ± 0.222 bB	29.85 ± 0.321 cC	30.71 ± 0.234 cB	32.32 ± 0.322 dA	36.19 ± 0.322 eB
	30	19.84± 0.492 aBC	25.44 ± 0.492 bB	28.66± 0.492 cBC	29.74 ±0.322 cAB	32.53 ± 0.492 dA	35.01± 0.492 eAB
	45	18.55 ± 0.492 aB	25.65 ± 0.492 bB	28.02± 0.186 cAB	29.41 ± 0.322 dA	31.78 ± 0.186 eA	34.68 ± 0.492 fA
	60	15.33 ± 0.201 aA	23.29 ± 0.322 bA	27.16 ± 0.322 cA	29.20 ± 0.186 dA	31.03 ± 0.234 eA	34.36 ± 0.265 fA
TBARS mg MDA/Kg	1	0.28 ± 0.006 fA	0.26 ± 0.007 eA	0.20 ± 0.001 dA	0.11 ± 0.004 cA	0.06 ± 0.006 bA	0.04 ± 0.004 aA
	15	0.36 ± 0.006 eB	0.28 ± 0.008 dA	0.22 ± 0.011 cA	0.13 ± 0.006 bB	0.09 ± 0.004 aB	0.07 ± 0.006 aB
	30	0.42 ± 0.011 fC	0.31 ± 0.007 eB	0.26 ± 0.004 dB	0.20 ± 0.008 cC	0.17 ± 0.003 bC	0.12 ± 0.004 aC
	45	0.58 ± 0.009 fD	0.49 ± 0.010 eC	0.41 ± 0.008 dC	0.36 ± 0.003 cD	0.29 ± 0.003 bD	0.20 ± 0.005 aD
	60	0.75 ± 0.007 fE	0.57 ± 0.005 cD	0.48 ± 0.008 dD	0.38 ± 0.003 cE	0.36 ± 0.003 bE	0.27 ± 0.005 aE

Emulsified meat system with nanoemulsion (EMSN), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and 2-thiobarbituric acid reactive substances (TBARS), Not detected (ND), gallic acid equivalents (GAE), ascorbic acid equivalents (AAE) and malonaldehyde (MDA). The lowercase letters in the superscript indicate significant differences (p < 0.05) between treatments (rows), and uppercase letters indicate significant differences in each treatment with respect to time (columns) (p < 0.05).

The results of the antioxidant activity (DPPH) assays exhibited significant differences (p < 0.05) between the treatments. The major activity was found in the treatment EMSN 5%; this activity was about 1.8-fold greater with respect to the EMSN 0% on day 60. The EMSN 0% exhibited antioxidant activity because the meat contains peptides with antioxidant properties, such as carnosine (β-alanyl-L-histidine) [35]. Sharma et al. [17] incorporated essential oils in chicken sausage and found major inhibition of DPPH radicals. The nanoemulsion contains xoconostle extracts and orange essential oil, which contain bioactive compounds, thus resulting in the increment in antioxidant activity (DPPH). The bioactive compounds inhibit free radicals [36,37,38]

The ABTS radical showed the same results as DPPH, with significant differences between the treatments (p < 0.05). Again, treatment EMSN 5% showed major antioxidant activity about 2.2-fold greater than the EMSN 0% (Table 4). Isaza et al. [31] found similar results after the incorporation of cherry extract in sausage. The phenols content and antioxidant activity were reduced with a controlled release during storage (Table 4). Again, treatments with nanoemulsions showed the best results.

Lipid oxidation showed significant differences (p < 0.05) between the treatments. Treatment EMSN 5% showed about a 2.7-fold reduction in the production of malonaldehyde (MDA) with respect to the EMSN 0%. Šojić et al. [39], Bianchin et al. [40], Erdmann et al. [41], and Ozogul et al. [42] found that the incorporation of essential oils in (their) meat systems reduced the production of malonaldehyde with respect to the control. The incorporation of the nanoemulsions with antioxidant compounds from xoconostle and orange essential oil delayed lipid oxidation, thus extending the shelf life of the EMSN.

4. Conclusions

The incorporation of the nanoemulsion in the emulsified meat system improved the nutritional contribution due to the increment in protein, inclusion of essential oils, and reduction in the loss of moisture. The texture profile showed increased hardness and chewiness. The bioactive compounds and antioxidant activities (DPPH and ABTS) incremented after the incorporation of the nanoemulsions, resulting in reduced production of malonaldehyde and minor lipid oxidation. The most favorable treatment was emulsified meat system with nanoemulsion (EMSN) 5%. Thus, the nanoemulsion extended the shelf life of the emulsified meat system.