Exploring the synergistic effects of essential oil and plant extract combinations to extend the shelf life and the sensory acceptance of meat products: multi-antioxidant systems

Abstract

To develop multi-antioxidant systems for the preservation of meat products, mixtures of essential oils or plant extracts were investigated for their antioxidant interactions. The combinatorial study revealed that the chemical diversity of both major and minor components of these ingredients contributed to the antioxidant interactions. A shift from antagonistic or additive interaction to synergistic one was achieved by modulating the ratio of mono-components of multi-antioxidant systems. Mixtures containing oregano/thyme (25/50 of IC50), thyme/clove (25/100) and thyme/cinnamon (50/25) oils as well as cranberry/rosemary (25/25), cranberry/green tea (25/25), cranberry/apple (25/25), rosemary/apple (50/25) and grapeseed/cranberry (50/50) extracts have shown synergistic antioxidant effects. Among the investigated systems, thyme/clove oils and oregano/thyme oils/grape-seed extract systems have extended, in situ, the shelf-life of chicken and ground pork products stored at 4 °C by 2 to 4 folds. The sensory acceptability of treated samples was rated to be moderately better than control. This study lays the ground for the development of efficient natural multi-antioxidant systems.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05653-4.

Introduction

The quality of meat products, during processing, cooking and storage, can be affected by the oxidation, which causes a deterioration of organoleptic (e.g. colour, flavor) properties and a loss of nutritional value leading to a reduced shelf life. The oxidation processes in meat can be related to lipids, proteins, and heme proteins and be initiated through metallic ions (e.g. endogenous heminic iron) and exogenous reactive oxygen species (Guyon et al. 2016). For instance, the oxidation of oxymyoglobin into metmyoglobin results in a brown color, and unpleasant flavours are generated due to oxidation of membrane phospholipids. Incorporating natural antioxidants, such as essential oils and plant extracts into refrigerated meat products, can enhance their shelf life; and this can be achieved through different methods including animal diets, mixed with meat products, onto the meat surface or active packaging. Indeed, these natural antioxidants were added to the meat in the range of 0.02 to 2%, and the oxidation decreases of 15–85% have been reported (Jiang and Xiong 2016). However, compared to synthetic preservatives, natural antioxidants can be limited by their strong contribution to the aroma, affecting the acceptance of the food products (Aziz and Karboune 2018; Nguyen et al. 2021). The hydrophilic nature of some natural antioxidants can limit their solubility in the hydrophobic medium and consequently reduces their potential use as antioxidants in high fatty food products (Karam et al. 2009).

Exploring the synergistic and additive interactions between natural antioxidants is a potential strategy to overcome their limitations and to design more potent multi-antioxidant systems containing lower amount of every single ingredient (Bounimi and Chebli 2017). However, as far as the authors are aware, no comprehensive study on the synergistic effects of natural antioxidant combinations has systematically investigated antioxidant efficiency, shelf-life extension, and sensory acceptance. In addition to essential oils containing volatile compounds such as phenols, allylic alcohols, and selected monoterpenes, various plant extracts have also shown the ability to inhibit lipid oxidation such as grape seed extract, lycopene from tomato and red pepper, carotenoids and tea catechins (Carpenter et al. 2007; Kulkarni et al. 2011; Lau and King 2003; Sasse et al. 2009). The presence of a wide variety of chemical compounds contributes to the antioxidant activity of plant extracts, for example polyphenols, anthocyanins and procyanidins in cranberry extract (Tamkutė et al. 2020), flavonoids in green tea extract (comprising 70% of its phenolic compounds) (Aziz and Karboune 2018), polyphenols, flavonoids and flavonols in apple extract (Li et al. 2020), phenolic diterpenes (e.g. carnosic acid, carnosol, rosemanol, epirosemanol, methyl carnosate), flavonoids (e.g. cirsimaritin, genkwanin) and phenolic acids (e.g. rosmarinic and caffeic acids) in rosemary extract and monomeric phenolic compounds (e.g. catechin, epicatechin, epicatechin- 3-O-gallate), and dimeric, trimeric and tetrameric procyanidins in the grape seed extract (Banon et al., 2007).

In our previous study (Khodaei et al. 2021), the chemical composition and antioxidant properties of thirty-eight essential oils were assessed. Among these essential oils, oregano, cinnamon, clove, pimento and thyme essential oils exhibited the highest antioxidant properties. These essential oils were selected in the present study to develop multi-antioxidant essential oil systems that synergistically act. In addition, five plant extracts, cranberry, apple, grapeseed, rosemary and green tea extracts were selected, and their combinations were also investigated in the present study with aim to identify those acting synergistically and develop multi-antioxidant plant extract systems. The efficiency of multi-antioxidant systems containing different proportions of essential oils and/or plant extracts in preventing meat oxidation were investigated. Finally, the sensory evaluation of meat products treated with these blends was conducted.

Materials and methods

Materials

List of plant extracts and essential oils and their source are presented in supplementary Table S1. Folin–Ciocalteau reagent (2 M with respect to acid), α-tocopherol (96%), 2,2-diphenyl-1-picrylhydrazyl (DPPH, > 90%), hexadecane (≥ 99%), C7-C40 saturated alkane standard (1000 μg/mL each component in hexane), hexane (≥ 97.0%), ethylenediaminetetraacetic acid (EDTA, ≥ 98%), butylated Hydroxytoluene (BHT, ≥ 99%), trichloroacetic acid (TCA, ≥ 99.0%), p-anisidine, malondialdehyde (MDA), sodium carbonate (≥ 99.5%), aluminum chloride (98%), gallic acid (≥ 98.0%), potassium chloride (99%), quercetin (≥ 95%), sodium acetate (> 99%), 4-dimethylaminocinnamaldehyde (DMAC, ≥ 98.0%),2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH, 97%), fluorescein sodium salt, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, 98%) were purchased from Millipore Sigma (St. Louis, MO, USA). Procyanidin A2 (> 99%) was obtained from Cedarlane Labs (Burlington, On). Acetone (≥ 99.5%) and methanol (≥ 99.9%) were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, United States) and anhydrous ethanol (≥ 99.9%) was purchased from Commercial Alcohols Inc. (Brampton, Ontario, Canada). Ultrapure water filtered with Milli-Q® system was used for all experiments. The water used in sensory analysis was drinking water.

The compositional analysis of the essential oils

Samples were analysed by gas chromatography/mass spectrometry using Agilent 7890A GC with a 7693 autosampler, a 5975C MSD, MSD ChemStation E.02.01.1777 software and a fused-silica capillary column coated with 5% diphenyl –95% dimethylsiloxane (Agilent J&W HP-5 ms GC column, 30 m, 0.25 mm, 0.25 µm). The temperature program included an increase of 3.0 °C/min from 50 to 155 °C and 10.0 °C/min from 155 to 250 °C with a 5 min post run at 300 °C. The injection volume was 1.0 µL, in the splitless mode and the carrier gas was helium (12 psi with average velocity of 43.535 cm/sec). The temperature of the inlet was 225 °C and the detector and interface were at 250 °C. Electronic impact (EI; 70 eV) ionization was used with ion source temperature of 230 °C. Acquisition mass range (m/z) was 40–500 Da with the scan rate of 5.92 scans/sec. Hexadecane (200 mg/L) was used spiked prior to GC/MS analysis and was used as internal standard for the quantification. Linear retention indices (LRI) were calculated according to Eq. 1 using C7-C40 saturated alkane standards in hexane (100–200 mg/L).

$$LRI=100\left(\frac{\text{ts - tn}}{\text{tn + 1 - tn}}+n\right)$$ 1

where t_s is retention time of target component, n is carbon number of preceding n-alkane and n + 1 is carbon number of subsequent n-alkane. The identification of samples was done using both LRI and Mass Spectral Database (NIST 11).

Essential oil samples were prepared in n-hexane. Quality control samples, consisted of a mixture of 4 essential oil samples (rosemary, pine, cypress, and verbena) spiked with the internal standard, were injected (every 5 injections) to monitor instrumental variations. Overall, the relative standard deviation (%RSD) observed for compounds in quality control samples ranged from 6.1 to 11.4%.

The compositional analysis of the plant extracts

Total phenolics content

Total phenolic content of the plant extracts was measured using Folin–Ciocalteau method (Ainsworth and Gillespie 2007). The extract solution was added to Folin–Ciocalteau reagent at a ratio of 1.6:0.1 (v/v). The mixture was vortexed thoroughly. Sodium carbonate solution (20%, 300 μL) was then added to the mixture (1.6 mL) before incubating it for 2 h at room temperature. The absorbance of the samples was recorded at 765 nm and the concentrations were calculated using a calibration curve using gallic acid standards (50, 100, 250, and 500 mg/L).

Total flavonoid content

Flavonoid content of the plant extracts was measured using the aluminum chloride colorimetric method (Kalita, Tapan, Pal, and Kalita, 2013). Samples (0.5 mL), methanol (1.5 mL), 1% (w/v) aluminum chloride solution (0.1 ml), 1 M potassium acetate (0.1 ml) and distilled water (2.8 ml) were mixed, and absorbance of the resulted solution was read at 415 nm. A calibration curve was constructed using quercetin (6.25–100 μg/mL).

Total anthocyanin content

The pH-differential method (Lee et al. 2005) was performed by reading the absorbance of the extracts at pH 1.0 (potassium chloride, 0.025 M) and pH 4.5 (sodium acetate, 0.4 M) at the λ_vis-max and at 700 nm. Anthocyanin content was calculated as cyanidin-3-glucoside equivalents (Eq. 2):

$$\text{Anthocyanin pigment}\left(\text{cyanidin}-3-\text{glucoside equivalents},\text{mg}/\text{L}\right)=\left(\text{A}\times \text{MW}\times \text{DF}\times 10^3\right)/\left(\epsilon \times \text{l}\right)$$ 2

where A = (A _λvis-max – A_700nm)_{pH 1.0} – (A _λvis-max – A_700nm)_{pH 4.5}, MW = molecular weight of cyanidin-3-glucoside, DF = dilution factor, l = pathlength in cm and ɛ = 26 900 molar extinction coefficient for cyanidin-3-glucoside (L × mol^–1 × cm^–1).

Total proanthocyanidins content

The total proanthocyanidins assay was done by measuring absorbance of the solution containing 280 μL of extracts and 840 μL of 0.1% (w/v) DMAC reagent in acidified ethanol at 640 nm. Procyanidin A2 (25–100 μg/mL in ethanol) was used as standard (Prior et al. 2010).

2-Diphenyl-1-picrylhydrazyl (DPPH) assay

Antioxidant activity of essential oils and plant extract was measured using DPPH assay (Prior et al. 2005). 50 μL of sample, 450 μL of 100 mM Tris–HCl buffer (pH 7.4), and 1 mL of 0.1 mM DPPH solutions were mixed and incubated in the dark for 30 min at room temperature. Absorbance at 517 nm was then measured using water as a blank. Positive and negative controls were analyzed similarly using α-tocopherol in ethanol (2 mg/mL) and methanol, respectively. The percentage of scavenging radical was calculated using the following Eq. 3:

$$\text{Scavenging radical}\left(\%\right)=\frac{A_{\mathit{Control}}-A_{\mathit{Sample}}}{A_{\mathit{Control}}}x100$$ 3

where A_Control is the absorbance of the control and A_Sample is the absorbance of the sample.

The inhibitory concentration (IC₅₀) corresponding to the concentration of samples required to inhibit 50% of the initial DPPH radicals was also estimated.

Oxygen radical absorption capacity (ORAC) assay

Modified method of Cao et al., (1993) was used for ORAC assay. different concentrations of essential oils or plant extracts in acetone/phosphate buffer (pH 7.4) and 16 mM fluorescein (prepared in PBS 10 mM pH 7.4) were pipetted into a 96-well black-walled plate and incubated at 37 °C for 30 min in a Synergy HTX Multi-Mode Reader. After addition of the AAPH (2,2’-azobis(2-amidinopropane) dihydrochloride, 79.65 mmol/L), fluorescence readings were taken for 1 h at 485 nm (excitation wavelength) and 520 nm (emission wavelength) every 60 s. The AUC (area under the curve) and Net AUC of the standards and samples were determined using Gen5 Data Analysis Software using the following Eqs (4, 5) respectively:

$$\text{AUC}=\left(\frac{R_1}{R_1}\right)+\left(\frac{R_2}{R_1}\right)+\left(\frac{R_3}{R_1}\right)+\dots +\left(\frac{R_n}{R_1}\right)$$ 4

where: R₁ is the fluorescence reading at the initiation of the reaction; R_n is the last measurement

$$\text{Net AUC}={\text{AUC}}_{\text{sample}}-{\text{AUC}}_{\text{blank}}$$ 5

Trolox ( ±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (100, 50, 25, 12.5, 6.25, and 0 mM) was used as a standard and the Trolox Equivalents (TE) of the samples calculated using the following formula:

$$\text{TE}\left(\text{range of concentrations}\right)={\text{m}}_{\text{compound}}/{\text{m}}_{\text{Trolox}}$$ 6

where: m_compound is the slope of the linear regression analysis of the compound; m_Trolox is the slope of the linear regression analysis of Trolox. Results are expressed as µmol Trolox Equivalents (TE)/g sample.

Fractional antioxidant capacity (FAC) index

To assess the effects of combining selected essential oils and extracts, the fractional antioxidant capacity index was estimated (Bounimi & Chebli 2017) for oils and extracts mixed at 25, 50, 75 and 100% percentage of IC₅₀ value. For instance, a 50/50 IC₅₀ mix of essential oil/extract corresponds to half of their IC50 concentration in the final antioxidant system. The index was determined by dividing the experimental value of the percentage of scavenging radical obtained by DPPH by the theoretical value. Theoretical values (Eq. 7) were obtained by considering the proportion of each essential oil and their IC₅₀ values when tested alone. The values were then categorized into antagonistic effect (FAC < 0.5), competitive effect (0.5 < FAC < 0.8), additive effect (0.8 < FAC < 1.0), or synergistic effect (FAC > 1.0).

$$\text{Fractional Antioxidant Concentration}\left(\text{FAC}\right)\text{index}=\frac{Experimentalvalue}{Theoreticalvalue\left[\left(Proportion1+Proportion2\right)\ast 0.5\right]}$$ 7

Effect of multi-antioxidant systems on chemical and sensory properties of the meat products

Shelf life of meat products was studied in terms of their oxidation; microbiological properties of products were not addressed in this study. Selected antioxidant mixes and their concentrations were identified based on the identified synergies and on preliminary experiments (data not shown). These mixes were used to make multi-antioxidant systems as per the following formulations:

• Formulation 1 (F1): 0.0312% (w/w) of oregano essential oil; 0.0312% (w/w) of thyme essential oil; 0.25% (w/w) of organic grape seed extract; 0.0312% (w/w) of emulsifier (Tween 80).

• Formulation 2 (F2): 0.00715% (w/w) of clove leaf essential oil; 0.0587% (w/w) of thyme essential oil; 0.0312% (w/w) of emulsifier (Tween 80).

• Formulation 3 (F3): 0.008% (w/w) of apple extract; 0.187% (w/w) of cranberry extract-02.

• Formulation 4 (F4): 0.0169% (w/w) of cranberry extract-01; 0.187% (w/w) of cranberry extract-02.

The efficiency of these multi-antioxidant systems to extent the shelf life of chicken and ground pork was assessed using accelerated and normal shelf-life tests.

Preparation of samples for shelf-life analysis

Chicken thighs were obtained from Exceldor, a local poultry company; the chickens were slaughtered on the day of delivery. Ground pork was obtained from Olymel, a local company and stored at 4 °C to maintain quality. For the accelerated shelf-life analysis, 5 chicken thighs without bones but with the skin were cold ground in order to avoid oxidation. Ground chicken or ground pork samples were then mixed with formulations (1%, w/w) from which 10 samples of 1.5 g were transferred to tubes for incubation at 35 °C for 48 h. Control samples were not treated with any of formulations and incubated at 35 °C for 48 h, while T0 samples were analysed without any incubation.

For the normal shelf-life test, the chicken thighs with bone and skin were sprayed with formulations to achieve a concentration of 1% (w/w). while the ground pork was mixed with formulations to achieve the concentration of 1% (w/w). The samples were then stored in polypropylene bag at 4 °C. At days 0, 6 and 17 of incubation, 3 chicken thighs were ground, and 9 samples of 1.5 g were taken to test for the presence of oxidation products using p-anisidine assay. for the ground pork, 6 samples (1.5 g) were analysed upon 0, 8 and 21 days of incubation.

Analysis of the oxidation products using p-anisidine assay

Meat samples of 1.5 g were mixed with 1 mL of 0.3% EDTA, 5 mL of 0.8% BHT in hexane and 8 ml of 0.3 M TCA, and vortexed for 2 min. BHT was added to avoid oxidation during analysis. After centrifugation for 5 min at 3500 × g (4 °C), the hexane phase was discarded and the TCA layer was recovered (Marcinčák et al. 2004). After addition of 200 µL of 0.25% (w/v) p-anisidine in acetic acid to 1 mL of sample, the absorbance was recorded at 350 nm after 10 min of incubation using MDA as a reference (Vilarinho et al. 2018).

Sensory evaluation of samples containing essential oils and extracts

Preparation of samples for sensory evaluation: Bone-in chicken thighs and ground pork were sprayed with mono- and multi-components antioxidant systems, including oregano essential oil and formulations (F1, ½F1) as follows:

• Formulation 1 (F1): 0.0312% (w/w) of oregano essential oil; 0.0312% (w/w) of thyme essential oil; 0.25% (w/w) of organic grape seed extract; 0.0312% (w/w) of emulsifier (Tween 80).

• Formulation ½F1: 0.0156% (w/w) of oregano essential oil; 0.0156% (w/w) of thyme essential oil; 0.125% (w/w) of organic grape seed extract; 0.0156% (w/w) of emulsifier (Tween 80).

The sprayed samples were placed in an airtight polypropylene storage bag and kept overnight at 4° C. After 24 h, the chicken thighs were cooked by steaming (25 min), and the ground pork were pan-fried (160°F, 25 min), and presented to panelists warm.

Consumer evaluation: A random block design was selected, and 40 untrained panelists (faculty, staff and students of McGill University) evaluated the samples containing mono- and multi-components antioxidants along with the control samples. For difference and rating tests, minimum number of 20–30 assessors are recommended (R. P. Carpenter, Lyon, and Hasdell, 2000). The panelists were asked to rate “overall” sensory properties of samples (e.g. texture, taste, aroma). The sensory attributes were evaluated using the Compusense software (Compusense Inc., Canada) with two hedonic scales: the first for degree of difference (1 = no difference, 5 = extremely different), and the second for degree of liking (1 = extremely worse than the control, 5 = extremely better than the control).

Results and discussion

Composition and antioxidant activity of multi-component essential oils and plant extracts

Table 1 summarizes the composition and the antioxidant activity (IC₅₀) of the five selected essential oils from ceylan cinnamon, clove leaf, oregano, pimento berry and thyme. The ceylan cinnamon, clove leaf and pimento berry essential oils composed of the highest content of phenols (786–888 mg/g oil) expressed the highest antioxidant activity with an IC₅₀ of 0.1 mg sample/mg DPPH. Contrary to the other investigated essential oils, oregano and thyme ones contained a higher content of monoterpenes (256.0 to 308.0 mg/g) and lower one of phenols (498.0–674.0 mg/g oil), which have reflected on a relatively lower antioxidant activity (IC₅₀ of 1 to 1.2 mg sample/mg DPPH). Moreover, sesquiterpenes are the second largest group in the clove leaf (94 mg/g) and pimento berry (84.0 mg/g) essential oils and esters are higher in Ceylan cinnamon essential oil (64 mg/g) as compared to other essential oils. Detailed composition (mg/g of oil) of selected essential oils is presented in Supplementary Table S2.

Table 1.

Composition and antioxidant properties of selected essential oils and plant extracts

Essential Oils
Compounds (mg/g of oil)	Ceylan cinnamon	Clove leaf	Oregano	Pimento berry	Thyme
Monoterpene	27.0 ± (1.6)		256.0 ± (42.2)	6.0 ± (0.05)	308.0 ± (25.3)
Sesquiterpene	34.0 ± (2.7)	94.0 ± (7.8)	10.0 ± (1.3)	84.0 ± (1.8)	11.0 ± (1.2)
Alcohol	19.0 ± (1.9)		21.0 ± (2.4)		68.0 ± (2.5)
Ketone		1.0 ± (0.06)			19.0 ± (1.0)
Phenol	786.0 ± (32.7)	796.0 ± (71.1)	674.0 ± (42.2)	888.0 ± (98.5)	498.0 ± (42.5)
Esters	64.0 ± (4.5)	1.0 ± (0.04)		8.0 ± (0. 6)
Other		9.0 ± (0.6)	3.0 ± (0.05)	8.0 ± (1.1)	3.0 ± (0.02)
IC50 (mg /mg DDPH)	0.12	0.13	1.0	0.10	1.17

Plant Extracts
Compounds	Cranberry extract-01	Cranberry extract-02	Apple extract	Grapeseed extract	Rosemary extract-01	Rosemary extract AquaRox15	Green tea extract
Total phenolic (g/100 g)	28.08 ± (2.06)	1.05 ± (0.05)	34.35 ± (0.75)	62.35 ± (13.58)	2.52 ± (0.41)	36.26 ± (2.63)	42.72 ± (5.80)
Flavonoid (g/100 g)	3.13 ± (0.30)	0.01 ± (0.00)	6.29 ± (0.14)	1.60 ± (0.18)		8.96 ± (0.59)	1.43 ± (0.14)
Anthocyanin (µg/g)	7.30 ± (2.28)	0.26 ± (0.00)	1.65 ± (0.00)	0.43 ± (0.03)		2.18 ± (0.09)
Procyanidin (mg/g)	1.02 ± (0.00)	8.11 ± (0.03)		1.03 ± (0.05)	20.47 ± (0.24)	1.09 ± (0.02)	2.61 ± (0.00)
IC50 (mg/mg DPPH)	0.76	8.43	0.36	0.14	0.78	0.52	0.19

On the other hand, antioxidant property of plant extracts can be attributed to their polyphenols, anthocyanins and/or procyanidins contents. The composition of the plant extracts, used in this study, is presented in Table 1. Green tea and grapeseed extracts showed the highest content in the total phenolic compounds of 42.72 and 62.35 g/100 g, respectively. Although cranberry extract-02 and rosemary extract are composed of lower content of total phenolic compounds (1.05 and 2.52 g/100 g, respectively), they showed higher procyanidin contents (8.11 and 20.47 mg/g, respectively). The compositions of the plant extracts in this study are comparable to those reported in literature. The total polyphenolic content of 28.7–31.1 g/100 g (dry matter) (Chamorro et al. 2012) and the procyanidin content of 0.5–1.9 g/100 g were reported for grapeseed extract (Gibis & Weiss 2012). On the other hand, the total polyphenolic content of aqueous extract, ethanolic extract and leaves of green tea has been reported to be 30.2, 83.7 and 20.5 g/100 g, respectively, which are falling within the range reported in this study (Gramza et al. 2006). The investigated cranberry extracts exhibited different phenolic enrichment levels (phenolic content of 1.05 and 28.08 g/100 g). Cranberries are known as a major source of procyanidins, and total polyphenols, anthocyanins and proanthocyanidins contents of 37.4, 3.3 and 10.0 g/100 g, respectively, have been reported for cranberry extract (Wallace & Giusti 2010). Moreover, Bubonja-Sonje et al (2011) reported that rosemary extract contains 45 g/100 g of polyphenols, and Bai et al. (2010), who optimized polyphenol extraction from apple pomace, found a polyphenol content of 4.4–6.2 g/100 g.

To assess the type of interactions (synergistic, additive, antagonistic) between the selected essential oils, the FAC index, expressed as the ratio of the experimental radical scavenging proportion and its respective simulation, was estimated. The identification of the multicomponent antioxidants with the highest synergistic interactions (FAC > 1.0) can allow improved antioxidant properties at the reduced concentrations of essential oils. Table 2 shows that synergistic effects were achieved with combinations of thyme/oregano (50/25 of IC₅₀), thyme/clove (25/100 of IC₅₀) and thyme/cinnamon (50/25 of IC₅₀). Eugenol was the major compound in ceylan cinnamon (785.9 mg/g), clove leaf (795.8 mg/g) and pimento berry (859.8 mg/g); however, combining these essential oils resulted in a competitive effect (0.5 < FAC < 0.8) in which adverse interactions were dominant. The use of ceylan cinnamon and pimento berry essential oils in excess in the combinations even promoted an antagonistic effect (FAC ≤ 0.5). For instance, ceylan cinnamon/clove leaf (75/25 of IC₅₀), pimento berry/ceylan cinnamon (75/25; 100/100 of IC₅₀) and pimento berry/ clove leaf (100/25 of IC₅₀) essential oil combinations led to a FAC of 0.2 to 0.5. Carvacrol and thymol were abundant in oregano (650.3 mg/g) and thyme (475.5 mg/g) essential oils, respectively (Table S2). Thyme oil also had relatively high content of monoterpenes (308.0 mg/g) with p-Cymene being the most abundant (203.7 mg/g). Synergistic effects were observed when thyme essential oil (IC₅₀ of 50%) was mixed with oregano (IC₅₀ of 25%) and cinnamon (IC₅₀ of 25%) ones. Increasing the proportion of thyme (IC₅₀ > 50%) and oregano (IC₅₀ > 25%) essential oils in their combinations favored an antagonistic interaction, which can be attributed to the changes in monoterpenes/phenol ratio. On the other hand, increasing the proportion of cinnamon (IC₅₀ > 25%) essential oil in the thyme/cinnamon combinations resulted in a competitive behavior (0.5 < FAC < 0.8). The results also indicate that thyme/clove leaf (25/100 of IC₅₀) essential oil combination showed a synergistic interaction. Although cinnamon and clove leaf essential oils are both rich in eugenol, they have shown different behaviors in combination with thyme essential oil. Contrary to cinnamon and clove leaf essential oils, pimento berry essential oil exhibited mainly an antagonistic effect when combined with thyme and oregano (Table 2). The results reveal that the contribution of the compositional diversity to the antioxidant activity may be more significant than the increase in the amount of the strongest antioxidant compounds. Moreover, minor molecules in essential oils may have also modulated the antioxidant activity of main components and, therefore, the observed synergistic or additive effects (Sharma et al. 2020). Thyme essential oil was used at higher ratio in a study where the combination between three essential oils were optimised using Simplex Lattice Mixture Design (FRAP and ABTS assay), and the highest synergistic effect was observed for a mixture containing 66.7% of Thymus vulgaris L., 16.7% of Coriandrum sativum L., and 16.7% of Apium graveolens L. (Crespo et al. 2019). Moreover, Bounimi and Chebli (2017), who tested three combinations of essential oils, reported that Thymus leptobotrys/Origanum compactum and Origanum compactum/Calamintha officinalis had synergistic effect, while Thymus leptobotrys/Calamintha officinalis had additive effect.

Table 2.

Effect of mixing essential oils on their antioxidant property (IC₅₀) expressed by the FAC index. The experimental DPPH values are presented as supplementary data (Table S3)

Similar to essential oils, combining plant extracts with different composition can lead to a complimentary effect in the antioxidant properties of the final mixture. Table 3 shows the FAC index of the combination of selected plant extracts at different proportions. As compared to essential oils, higher number of the plant extract combinations showed synergistic effect. Cranberry extract-01, rosemary extract AquaRox15 and rosemary extract led to the combinations with the highest synergistic interactions (FAC > 1–1.5). Rosemary extract-01 had relatively low polyphenol content (2.52% w/w) and moderate antioxidant property (IC₅₀ of 0.8 mg sample/mg DPPH), while rosemary extract AquaRox15 had relatively high polyphenol content (36.26% w/w) and antioxidant property (IC₅₀ of 0.5 mg sample/mg DPPH). This reveals that the observed synergy is independent on the initial antioxidant strength. In addition, grapeseed extract and apple extract (IC₅₀ of 25–50%) showed a synergistic effect with cranberry extract-01 and rosemary extract AquaRox15. However, combining grapeseed and apple extracts led to antagonistic interactions. The lowest total polyphenolic content of cranberry extract-02 are reflected on their low antioxidant activity, which was competitive or antagonistic with all extracts; expect with cranberry extract 1 (IC₅₀ of 100%), apple extract (IC₅₀ of 25–75%) and rosemary extract AquaRox15 (IC₅₀ of 75–100%), a synergistic effect was observed.

Table 3.

Effect of mixing extracts on their antioxidant property (IC₅₀) expressed by the FAC index. The experimental DPPH values are presented as supplementary data (Table S4).

The combinations of essential oils or plant extracts were also assessed using hydrogen atom transfer (HAT)-based ORAC assay (Table 4). In our study, phenols paired with esters and alcohols showed synergistic effects on hydrogen atom transfer-based ORAC value, whereas the opposite was true on single electron transfer-based IC₅₀ when paired with monoterpenes and ketone (Khodaei et al. 2021). Table 4 shows that higher number of combinations for both essential oils and extracts showed synergistic effect when hydrogen atom transfer-based ORAC value was used instead of single electron transfer-based IC₅₀. The difference in observed results may arise from steric hindrance reducing the access of phenols to the DPPH radical site. Reaction is rapid with simple phenols with no ring adduct and but is slow with higher number of acid groups or side chains connected to aromatic rings (Schaich et al. 2015). Compound’s reduction potential can affect the result of ORAC assay since it is based on the transfer of hydrogen atom and electron. While mixtures of compounds with similar reduction potential lowers antioxidant properties, and adding flavonoid with lower reduction potential to other flavonoids can improve ORAC value (Freeman et al. 2010).

Table 4.

Comparison of FAC index for essential oils or plant extract mixes (50/50 percentage of IC50 value) using ORAC and DPPH assays

Shelf-life analysis of meat products treated with selected multi-antioxidant systems

Based on the ORAC and DPPH results, the following combinations, clove/thyme essential oils (F2), apple /cranberry-02 polyphenol extracts (F3) and cranberry-01/cranberry-02 polyphenol extracts (F4) were selected as multi-antioxidant systems for assessing their antioxidant efficiency in situ using meat products. In addition, the mix of oregano essential oil, thyme essential oil and grape seed extract (F1) was also evaluated as it was identified as the best combinations for the antimicrobial activity in our previous study (Bagheri et al. 2020). Grape seed extract has been used at different concentrations (0.005–2%) in beef, pork and poultry meat products and resulted in 0.3 to tenfold decrease in oxidation (Carpenter et al. 2007; Kulkarni et al. 2011; Lau & King 2003; Sasse et al. 2009).

During the first stage of lipid oxidation, the formation of lipid hydroperoxides and conjugated dienes/trienes occurs; these compounds are considered as primary oxidation products. Rancidity and off-flavour of food products are mostly caused by the secondary lipid oxidation products, such as carbonyl, aldehydes, volatiles and malondialdehyde compounds, which are generated after the decomposition of hydroperoxides. The degree of the oxidation of the meat products was estimated through the quantification of secondary oxidation products using p‐anisidine assay (Lund et al. 2011). Figure 1 shows the amount of oxidation products (expressed as equivalent of MDA, µg/kg of meat) formed upon the incubation of chicken and ground pork meat products.

Fig. 1.

Oxidation of chicken and ground pork at the presence of essential oils/extract formulations using accelerated shelf life (35 °C, 48 h) and normal shelf life (4 °C) analysis. Formulation 1 (F1): 0.0312% (w/w) of oregano essential oil; 0.0312% (w/w) of thyme essential oil; 0.25% (w/w) of organic grape seed extract; 0.0312% (w/w) of emulsifier (Tween 80). Formulation 2 (F2): 0.00715% (w/w) of clove leaf essential oil; 0.0587% (w/w) of thyme essential oil; 0.0312% (w/w) of emulsifier (Tween 80). Formulation 3 (F3): 0.008% (w/w) of apple extract; 0.187% (w/w) of cranberry extract-02. Formulation 4 (F4): 0.0169% (w/w) of cranberry extract-01; 0.187% (w/w) of cranberry extract-02. 6 replicates of ground pork and 9 replicates of chicken for each formulation and control

The accelerated shelf-life test of chicken (Fig. 1A) shows that after 48 h of incubation at 35 °C, chicken treated with F1, F2 and control had 100.7% (3058.3 µg/kg), 109.9% (3199.0 µg/kg) and 178.1% (4238.5 µg/kg) increase in the amount of oxidation products, respectively as compared to day 0. The oxidation occurring in samples treated with F1 (p-value of 0.00196 and t-value of 5.05406) and F2 (p-value of 0.00577 and t-value of 4.07356) was significantly lower than that of the control. In contrast, F3 (6199.5 µg/kg) and F4 (4438.4 µg/kg)-treated chicken samples did not inhibit oxidation as compared to control. The observed results could be attributed to the fact that antioxidant compounds can be found in free and bound forms (e.g. covalently and ionically bound), which can affect their diffusion in food system. For instance, phenolic acids can be in the insoluble bound form. Moreover, flavonoids can be present in the glycoside form with sugar substitution, resulting in steric hinderance. The amount of oxidation products in control sample is close to the threshold value (5 mg MDA/kg meat) for detectable off-odour measured using thiobarbituric acid (TBARS) assay (Insausti et al. 2001). The contributions of the microbial growth and the antimicrobial properties of the formulations to the results of the accelerated shelf-life test of chicken have to be acknowledged. Therefore, normal shelf-life analysis of chicken was carried out to confirm the efficiency of the F1 and F2 formulations.

Normal shelf-life analysis of chicken (Fig. 1B) showed the oxidation rates of samples treated with F1 and F2 formulations were not significantly different from the control after 1 day (F-value of 0.053963 and p-value of 0.947695) and 6 days (F-value of 0.374035 and p-value of 0.694624). After 17 days of incubation, oxidation products increased to 369.0, 89.1 and 81.3% of their amount on day 1 for control (14,528.1 µg/kg), F1 (5545.1 µg/kg) and F2 (5317.3 µg/kg) treated samples respectively. These results confirm that antioxidant components of essential oils retained their activity during the course of incubation resulting in significant protection achieved by F1 (t-value of 8.6175 and p-value of 0.000498) and F2 (t-value of 8.15919 and p-value of 0.000091)-treated chickens against oxidation in prolonged storage as compared to control. Fratianni et al. (2010) have dipped chicken pieces in agar solution containing 0.5% balm and thyme essential oils and measured DPPH scavenging activity during 3-week storage at 4 °C and reported that thyme and, to a lesser extent, balm maintained their ability to inhibit the DPPH radical formation during the storage.

Both accelerated and normal shelf life analysis showed that F1 and F2 have similar capacity at protecting chicken against oxidation. Chouliara et al. (2007) have reported that the treatment of chicken with 0.1% and 1% (w/w) of oregano oil did not significantly affect the degree of lipid oxidation upon 9 days (TBARS value of 0.24–0.82 mg MDA/kg meat). In contrast, grape seed extract at a concentration of 1.6 g/kg reduced the oxidation in cooked turkey breast stored for 13 days (TBARS values of 0.50–4.20 mg/kg) (Mielnik et al. 2006).

Figure 1C shows the results of accelerated shelf-life analysis for ground pork. The control samples (3703.0 µg/kg) exhibited 46.9% increase in the oxidation after incubation. The results also show that there was no significant increase in the oxidation for F1, F2, F3 and F4-treated ground pork samples (2541.2, 2569.4, 2011.0 and 2211.0 µg/kg, respectively) as compared to time 0 (F-value of 0.3805 and p-value of 0.8194). These results reveal that the essential oil combination had the same efficiency as the plant extract mixtures in preventing oxidation. Figure 1D shows the amount of the oxidation of products during normal shelf-life analysis. No significant difference was observed between control, F1, F2 and F3-treated ground pork samples (2829.8, 2494.9, 2916.7 and 2808.0 µg/kg, respectively) on day 1 (F-value of 0.001137 and p-value of 0.999944) and on day 8 of incubation (4298.8, 2768.6, 4166.8 and 3119.0 µg/kg, respectively; F-value of 0.514602 and p-value of 0.607918). At longer incubation time of 21 days, F1 (108.5%, 5202.5 µg/kg, t-value of 3.39464 and p-value of 0.005763) and F3 (66.3%, 4669.0 µg/kg, t-value of 4.6854 and p-value of 0.0011) formulations improved stability of samples against oxidation as compared to control (270.6%, 10,487.7), while the effect of F2 was not significantly different from control (170.8%, 7686.8 µg/kg, t-value of 1.76063 and p-value of 0.058168). Therefore, it can be concluded that unlike chicken, the use of F1 and F3 formulations led to a better protection against oxidation for ground pork. The potential of grape seed extract at reducing hexanal formation (TBARS assay) in cooked and stored ground beef has been reported to be higher than BHA/BHT and similar to pine bark extract, and oleoresin rosemary (Ahn et al. 2007). Moreover, blackcurrant extract has been reported to reduce oxidation in pork patties after 9 days of incubation at 4 °C (Jia et al. 2012). In contrast, the use of thyme essential oil alone (0.1%, v/w) did not protect lamb meat from oxidation within the 21-day of incubation aerobically at 4 °C (1.4 to 3.5 mg MDA/kg meat). In another study, extracts from tea, grape, chestnut and seaweed were evaluated in pork patties during 20 days of storage in modified atmosphere packs at 2 °C, and grape and green tea extracts were the most effective antioxidants (Lorenzo et al. 2014).

Sensory analysis of meat products containing selected ingredients

Figures 2A and B show the sensory properties of chicken sprayed with 0.005 and 0.025% (w/w) oregano essential oil or essential oil/extract blends (F1 and ½F1) compared to the control. A larger percentage of panelists evaluated that chicken treated with oregano essential oil at 0.005% (w/w) was “very different” compared to the control (Fig. 2A, 53.3%, P value < 0.0001), and was rated as “moderately better than the control” (Fig. 2B, 66.7%, P value < 0.0001). At the 0.025% oregano essential oil concentration, equal number of panelists (31.3%) found the treated sample to be “moderately different”, “very different” and “extremely different”. The chicken treated with oregano essential oil at 0.025% (w/w) was favorably rated in terms of liking by the largest group of panelists (P value < 0.0001), with 62.5% stating it was “moderately better than the control” (P value < 0.0001). For ½F1-treated chicken sample, it was very well perceived by panelists, as 37.8% did not detect any difference compared to the control and 43.2% stated that the samples were “slightly different” from control (P value < 0.0001). While for the F1-treated chicken sample, 42.4% of panelists found that it was either “moderately” or “very” different compared to the control. In terms of liking, larger proportions of panelists (F1, 65%, p value < 0.00001; ½F1, 42.5%, p value < 0.00001) evaluated the F1 and ½F1-treated chicken samples as being “moderately better than the control”, most likely due to the additional seasoning.

Fig. 2.

Difference A and B and liking C and D of chicken and pork sausage samples treated with essential oils/extract blends and oregano essential oil as compared to control (40 panelists). Formulation 1 (F1): 0.0312% (w/w) of oregano essential oil; 0.0312% (w/w) of thyme essential oil; 0.25% (w/w) of organic grape seed extract; 0.0312% (w/w) of emulsifier (Tween 80). Formulation ½F1: 0.0156% (w/w) of oregano essential oil; 0.0156% (w/w) of thyme essential oil; 0.125% (w/w) of organic grape seed extract; 0.0156% (w/w) of emulsifier (Tween 80)

The ground pork samples showed different results as compared to chicken (Fig. 2C and D). 46.7% of panelists found “no difference” between ground pork sample treated with 0.005% (w/w) oregano essential oil and the control one, and the threated sample was rated as “moderately better than the control” in terms of liking by 76.7% panelists. The use of higher concentration of oregano essential oil (0.025%, w/w) led to more or less to the same proportion of panelists (43.4%), who have detected “no difference” compared to the control (p value < 0.0001), while 63.3% of panelist preferred these treated samples as “Moderately better than the control” (p value < 0.0001). In comparison, Karabagias et al. (2011) reported thyme and oregano essential oils concentration of more than 0.3% (v/w) in lamb to be strong and unacceptable. In another study conducted by Solomakos et al. (2008), the organoleptic properties of minced beef containing 0.3% and 0.6% (v/w) thyme essential oil were acceptable by the panelists whereas at 0.9% the product was rejected. In terms of blended samples, 45.7% of panelists detected a moderate difference between the F1-treated and control samples, while only a slight difference was noted by 40% of panelists with the ½F1 treated ground pork. Similar to the chicken, more than half of panelists preferred the ½F1-treated ground pork sample, while 37.1% did not observe a difference with the ½F1-treated sample in terms of liking.

Conclusions

The investigation of the interactions between selected essential oils or plant extracts revealed no direct correlation between the concentration of major compounds and the antioxidant properties in the mixtures. Indeed, the chemical diversity of the multi-antioxidant systems and the contribution of minor compounds seemed to be important. Shifting antagonistic or additive effects into synergistic effects requires a modulation of the ratio of the combinations of essential oils or plant extracts. The in-situ application of oregano oil/thyme oil/grape extract mixtures (F1) and clove/thyme essential oils (F2) reduced oxidation in chicken and ground pork in accelerated and normal shelf life as compared to control (1.6 – 4.5-fold reduction). The use of apple polyphenol/cranberry extracts (F3) as a multi-antioxidant system protected the ground pork product against oxidation, but not the chicken product. The sensory analysis showed that 62.2 and 97.3% of panelists perceived no, slight or moderate difference between F1 or ½F1-treated chickens and the control, respectively; while for ground pork product, more than 90% of panelists have no, slight or moderate difference for both formulations. Moreover, both F1-treated chicken and ground pork products were preferred in terms of likeness compared to controls with no ingredient.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

DPPH

2,2-Diphenyl-1-picrylhydrazyl

EDTA

Ethylenediaminetetraacetic acid

BHT

Butylated hydroxytoluene

TCA

Trichloroacetic acid

MDA

Malondialdehyde

DMAC

4-Dimethylamino-cinnamaldehyde

AAPH

2,2′-Azobis(2-methylpropionamidine) dihydrochloride

LRI

Linear retention indices,

FAC

Fractional antioxidant capacity

RSD

Relative standard deviation, %

IC50

Inhibitory concentration

ORAC

Oxygen radical absorption capacity

Author Contribution

NK conceived, validated the methodology, carried out the experiments, wrote the first draft of manuscript and corrected the manuscript; MH validated the methodology, carried out the experiments and contributed to the editing of the manuscript; SB conceived, contributed to the analyses and the validation of data and edited the manuscript; SK secured the funding, supervised the work, validated the methodology and the data analyses and edited the manuscript.

Funding

This study was supported by the Ministère de l’Agriculture, des Pêcheries, et de l’Alimentation du Québec (MAPAQ), through the Consortium de Recherche Innovation Transformation Alimentaire (RITA).

Availability of Data Material

All data generated or analysed during this study are included in this published article and its and its supplementary information files.

Code availability

Not applicable.

Declarations

Conflict of Interest

Not applicable.

Consent to participate

Not applicable”.

Consent for publication

Not applicable”.

Ethics approval

Not applicable”.