Effects of Cuminum cyminum L. essential oil and its nanoemulsion on oxidative stability and microbial growth in mayonnaise during storage

Abstract The present study aimed to investigate the effects of Cuminum cyminum L. essential oil (CEO) and its nanoemulsion (CEON) on oxidative stability and microbial growth of mayonnaise during storage. The GC analysis indicated that Cuminaldehyde (27.99%), o‐Cymene (17.31%), γ‐Terpinen (16.67%), and β‐Pinene (9.35%) were the major components of CEO, respectively. The assessments of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) showed that Escherichia coli ATCC 25922 (MBCCEO = 12 and MBCCEON = 6 mg/mL) was the most resistant bacteria, and in contrast, Staphylococcus aureus ATCC 29213 (MBCCEO = 6 and MBCCEON = 3 mg/mL) was the most sensitive bacteria. In the radical‐scavenging assay, CEON (IC50 = 5 ± 0.07 μg/mL) exhibited a higher antioxidant activity than CEO (IC50 = 10 ± 0.13 μg/mL). The results showed that applying the MBC of CEO and CEON in mayonnaise led to a significant decrease (p < .05) in acidity, peroxide value, number of acid‐resistant bacteria and fungi, and total microbial count compared with the control sample. In conclusion, this study demonstrated that using CEON resulted in oxidative stability, microbial growth control, and desirable sensorial attributes in mayonnaise compared with CEO and control samples.

oxidative damage through the deactivation of free radicals, prooxidants suppression, inhibition of the activity of enzymes producer free radicals, the boosting of the function of antioxidant enzymes, and oxidation control (Kishk & Elsheshetawy, 2013;Lu et al., 2010;Sørensen et al., 2017). In recent decades, synthetic antioxidants including butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been used to retard lipid oxidation due to their high effectiveness, affordable production, and desirable oxidative stability in comparison with natural antioxidants (Li et al., 2014). In contrast, synthetic antioxidants have disadvantages including low water solubility, safety concerns, and health issues such as gastrointestinal tract problems, fatty liver, skin allergies, storage in adipose tissue, and carcinogenesis (Gülçin, 2002;Lourenco et al., 2019;Valenzuela & Nieto, 1996). Recently, due to the side effects of synthetic antioxidants, there has been a high tendency to use natural antioxidants, as a suitable alternative, especially those with herb and spice origins (Alizadeh et al., 2019). Cuminum cyminum L. is a species of the Apiaceae family and indigenous to Southwest Asia and the Eastern Mediterranean countries. In many countries, cumin is widely used as an aromatic plant and spice for flavoring foods (Mandal & DebMandal, 2016). Scientific reports have shown that Cuminum cyminum L. and its essential oil as a natural food preservative possess antioxidant, antimicrobial, antifungal, and therapeutic properties (De et al., 2003;Mandal & DebMandal, 2016). The antioxidant activity of Cuminum cyminum L. essential oil (CEO) can be related to the existence of phenolic and polyphenolic compounds.
Nanoemulsions as kinetically stable colloidal systems with droplet size between 20 nm and 200 nm (Lago et al., 2019;Li & Chiang, 2012) possess functional properties and resistance to gravitational separation, coalescence, and aggregation in comparison with conventional emulsions (McClements, 2011). Essential oils nanoemulsions can be used more effectively in the food industry due to the larger contact area between bioactive compounds and food matrix than pure essential oils; consequently, their antioxidant and antimicrobial activities increase (McClements, 2011;Otoni et al., 2014). The first objective of the present study was to determine the antioxidant and antimicrobial activities of the CEO and Cuminum cyminum L. essential oil nanoemulsions (CEON) against food-borne microorganisms.
The second objective was to evaluate the microbial growth and oxidative stability of mayonnaise containing CEO and CEON compared with control during storage.

| Preparation of Cuminum cyminum L. essential oil
Cuminum cyminum L. essential oil from the Northeast region of Iran was supplied by Johare-Taem company and stored in a dry, dark, and cool place (Mashhad, Iran).

| Gas chromatography-mass spectrometry analysis
The chemical composition analysis of CEO was performed by gas chromatography-mass spectrometry (GC-MS) model Scion-456-SQ-Netherlands (Scion, UK) using HP-5MS capillary column, CP Sil 5 (25 m, 0.25 mm, film thickness 0.25 μm). The GC was performed at the injector temperature of 250°C, split 100 with the following conditions: helium gas as the carrier gas with a flow rate of 1 mL/min; at first, the column temperature was held at 45°C for 2 min then increased to 220°C at rate 3°C/min (hold for 5 min) and lastly to 270°C at rate 15°C/min (hold for 5 min); volume injected, 1 μL of the oil; and split ratio, 1:100. The MS operating parameters were as follows: electron energy 70 eV; source temperature 230°C; transfer line temperature 230°C; mass up to 650 resolution 0.7. The components of the CEO were identified using the retention time data from the National Institute of Standards and Technology (NIST) data collection (Kabouche et al., 2009;Sharifi et al., 2021).

| Preparation of CEO and CEON
Cuminum cyminum L. essential oil nanoemulsions was prepared using the methods described by Gahruie et al. (2017) and Chu et al. (2020) at ambient temperature (approximately 22°C). CEO (disperse phase) and deionized water (continuous phase) were used to make CEON.
The obtained mixture was homogenized at 12,000 rpm for 4 min using a laboratory stirrer (OS20-Pro, Dragon, China). Then, the coarse emulsion was exposed to ultrasonic for 10 min using a homogenizer in ultrasonic (probe) technology (BANDELIN SONOPULS HD 3100, BANDELIN, Germany) with a 30% power output of 225 W and 25 kHz.

| Particle size measurement
The average droplet size (z-average) of nanoemulsions and polydispersity index (PDI) were determined using a size analyzer model Nano-ZS90 (Malvern, UK) at 25°C. First, CEON was diluted with deionized water to 10:1000 to avoid multiple scattering problems.
The span of emulsion droplet sizes was measured using the following Equation (1). where D90, D50, and D10 are particle sizes of CEON corresponding to 90, 50, and 10% intensity on a relative cumulative particle size distribution curve.

| Scanning electron microscope of CEON
The morphology of CEON was characterized using a scanning electron microscope (SEM) model Quanta 450 FEG (FEI, USA). CEON were lyophilized, then sputtered with a thin layer of gold. SEM images were taken at an operating voltage of 30.0 kV.

| Antioxidant activity assay
The efficacy of the CEO and CEON to scavenge 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radicals was determined (Cuendet et al., 1997;Kirby & Schmidt, 1997). First, 25 μL of different dilutions (0.01%, 0.1%, and 1%) of each of CEO and CEON was mixed with 2.5 mL of 0.004% methanol solution of DPPH and subsequently, incubated for 30 min at ambient temperature (approximately 22°C). The absorbance of samples was measured against the control using spectrophotometry at 517 nm. The inhibition percentage was determined using Equation (2). AC = absorbance of the control (containing all reagents except the test compound); AS = absorbance of the sample.
The IC 50 (μg/mL) is the concentration of antioxidant required for 50% DPPH free radical scavenging.

| Antibacterial activity assay of CEO and CEON
The antibacterial activity of CEON and CEO was evaluated against

| Time-kill kinetic analysis of CEO and CEON
The inhibitory effects of CEO and CEON on the growth curve of E. coli ATCC 25922 and S. aureus ATCC 29213, as the most resistant and sensitive bacteria respectively, were investigated (Avila et al., 1999). MHB was inoculated with test bacteria at 1% (v/v), and then treated with CEO and CEON at ½ MIC, followed by incubation at 37°C. The control contains DMSO 1% (v/v) instead of CEO and CEON. The optical density of microbial supernatants at 600 nm was measured by UV-VIS spectrophotometer for 9 h at 0.5 h intervals.
Carboxymethyl cellulose, starch, and xanthan gum were mixed with oil and homogenized for 2 min/10,000 rpm. Then, this mixture and vinegar were slowly added to the aqueous phase and homogenized (8 min/1000 rpm) for emulsion formation. To prepare the treated samples, CEO (12 mg/mL) and CEON (6 mg/mL) were dissolved into the soybean oil and added to the mentioned recipe. The concentrations of CEO and CEON were chosen based on the results of antimicrobial (MBC) and antioxidant (DPPH radical-scavenging assay) activities, then mayonnaises were distributed into small glass jars (200 gr). The jars, after sealing and labeling, were stored at ambient temperature (approximately 22°C) for 3 months. The mayonnaises were aseptically sampled at four-time intervals (0, 1, 2, and 3 months) during storage for further analyses.

| Determination of peroxide value
The peroxide value (PV) of mayonnaise samples was determined by iodometric titration according to Bligh and Dyer (1959) with slight modifications. For oil extraction from mayonnaise, 10 g of sample was mixed with 20 mL methanol and 10 mL chloroform for 2 min, then centrifuged at 2000 rpm for 10 min. The oil phase was separated, and residual solvents were removed using a rotary at 60°C. For peroxide assay, 1 g of potassium iodide was added to 1 g of extracted oil and then mixed with 20 mL solvent containing chloroform and acetic acid (2:3 ratio). After boiling the mixture for 30 s, 50 mL distilled water and 20 mL potassium iodide 5% were added, and then it was titrated by sodium sulfate (1/500 N) in the presence of starch solution. The peroxide value was measured as Equation (3).
where V is the volume of expended sodium sulfate, N is the normality of sodium sulfate, and M is the sample weight.

| Acid value (AV) assay
For AV determination, the oil dissolved in ethanol/chloroform was titrated with 0.1 N sodium hydroxide in the presence of phenolphthalein as an indicator, according to AOCS (1997). The AV was calculated as Equation (4).
where V is the volume of expended NaOH, N is the normality of NaOH, and W is the analyte weight.

| Sensory analysis
The sensory properties of mayonnaise samples were evaluated at the end of 3 months of storage. The samples were assessed for color, taste, odor, texture, and overall acceptability based on the 5-point hedonic scale (1 = least acceptable, 5 = most acceptable).

| Statistical analysis
Statistical analysis was carried out by ANOVA (p < .05) using the SPSS, version 16. The significant differences between means were compared using the LSD test. All experiments were performed in triplicate.  (2004)

| Droplet size and microstructure of CEON
In general, reducing the particle size leads to an increase in the surface area of particles (Csicsák et al., 2023), cumin essential oil (Rostami et al., 2018), and cumin seed oil (Farshi et al., 2017), respectively. Additionally, the droplet size of thyme essential oil nanoemulsion was reported to be 82.5-125.5 nm (Xue et al., 2015).

| Antioxidant activity of CEO and CEON
The scavenging effects of CEO and CEON against DPPH radical were determined. According to our results, CEON exhibited stronger antioxidant activity with IC 50 = 5 ± 0.07 (μg/mL) compared with CEO with IC 50 = 10 ± 0.13 (μg/mL). The stronger antioxidant activity of the essential oil nanoemulsions can be related to the smaller size, greater solubility, and better permeability of droplets than essential oil emulsions; as a result, more free radicals are involved by the scavenging effects of essential oil nanoemulsions. Moreover, the pure essential oil is not able to dissolve in aqueous systems (Dhifi et al., 2016), which reduces its antioxidant activity compared with the nanoemulsion essential oil (Sharifi & Sharifi, 2023). However, nanoemulsions can dissolve in aqueous systems, which leads to the efficient release of activated compounds; subsequently, they can more effectively scavenge radicals (Lou et al., 2017). In previous studies,  indicated that essential oils are slightly more effective against grampositive than gram-negative bacteria (Burt, 2004;Chao et al., 2000;Mumivand et al., 2019). Gram-negative bacteria are less sensitive to the inhibitory effect of essential oils due to an outer membrane in their cell wall (Ratledge & Wilkinson, 1988), which limits the distribution of hydrophobic compounds throughout the hydrophilic layer (Vaara, 1992). As cited in previous studies, the Cuminum cyminum L. Abbreviations: CEO, Cuminum cyminum L. essential oil; CEON, Cuminum cyminum L. essential oil nanoemulsion.

TA B L E 2 Minimum inhibitory concentrations (MIC) and Minimum
Bactericidal Concentration (MBC) of CEO and CEON against food-borne bacteria.

F I G U R E 2
The growth curves of E. coli (a) and S. aureus (b) affected by CEO and CEON at ½ MIC in comparison with the control. CEO, Cuminum cyminum L. essential oil; CEON, Cuminum cyminum L. essential oil nanoemulsion; MIC, Minimum inhibitory concentration.
The high antimicrobial activity of CEO and CEON can be attributed to their major compounds, including Cuminaldehyde (Ghannay et al., 2022;Ghiasi et al., 2021;Wongkattiya et al., 2019), Terpinen (Bordini et al., 2018), and β-Pinene (da Silva Rivas et al., 2012;Salehi et al., 2019). According to da Silva Rivas et al. (2012), pinenes, as one of the major components of CEO, can inhibit the activity of esterase and phospholipase of microorganisms. Furthermore, the results indicated that the inhibitory effect of nanoemulsified essential oil (CEON) was higher than that of pure essential oil (CEO). The sensitivity of test bacteria, especially S. aureus, to CEON is due to the fusion of the cellular lipid membranes with essential oils (Valgas et al., 2007). Zhang et al. (2009) reported that CEON leads to an increase in cytoplasmic leakage from pathogenic cells compared with the CEO. The fusion of CEON with the cellular lipid membrane leads to the degradation of cell membrane integrity; subsequently, the membrane permeability due to the destabilization of cellular structure causes increasing cytoplasmic leakage, and consequently cell death (Baker Jr et al., 2003).

| Effect of CEON and CEO on peroxide value (PV)
The PV is measured to assess the primary oxidation of lipids by determining the concentration of hydroperoxides and peroxides.
The results showed that the PVs of three treatments significantly (p < .05) increased during storage (Figure 3). In the third month, and butylated hydroxytoluene (BHT) (Gavahian et al., 2013). The antioxidant activity of essential oils can be due to the presence of phenolic compounds in these products, which react directly with free radicals resulting from the first stages of lipid oxidation (Gavahian et al., 2013;Guillén & Cabo, 2002). Kwon et al. (2015) indicated that tocopherol, as a natural antioxidant, could reduce PV from 7.84 to 2.30 meq O 2 /kg oil in mayonnaise during storage.
According to Kong and Singh (2011), PV should not exceed 10-20 (meq O 2 /kg oil) to avoid the rancid taste of food products containing F I G U R E 3 Effect of CEON and CEO on PV (meq O 2 /kg oil) of mayonnaise during 90 days of storage. Vertical bars represent the standard deviation (n = 3). Different letters indicate statistically significant differences (p < .05). CEO, Cuminum cyminum L. essential oil; CEON, Cuminum cyminum L. essential oil nanoemulsion.
oil. In the current study, PV increased in all samples during 3 months of storage. Therefore, the shelf life of mayonnaise containing CEO and CEON is not recommended to exceed 3 months.

| Effect of CEON and CEO on acid value
The acid value of oil extracted from mayonnaise is referred to the presence of carboxylic acid groups in fatty acids. The production of free fatty acids may be due to the breakdown of the ester groups of chemical compositions and then converting them into acidic compounds, oxidative reactions, hydrolysis of triglycerides, and microbial activity in the presence of water (Andres et al., 2005;Gavahian et al., 2013;Stephen & Phillips, 2016). As shown in Figure 4 the AVs of all samples significantly (p < .05) increased during 3 months of storage; however, the AVs in CEON and CEO were lower than the control. Similarly, the increasing trend of AV in mayonnaise was reported during the storage period according to Kishk and Elsheshetawy (2013) and Alizadeh et al. (2019). Alizadeh et al. (2019) indicated that among the mayonnaise samples containing natural and synthetic antioxidants (tertiary butylhydroquinone), adding rosemary essential oil led to the lowest AV.
The increase in AV can be related to the breakdown of iron connections with phosvitin in low pH during the storage period; consequently, released iron activates oxidative and hydrolytic reactions by enzymes in eggs (Honold et al., 2016;Kishk & Elsheshetawy, 2013). On the other hand, microbial activities in the nutrient matrix of mayonnaise, because of the nonthermal process, lead to organic acid production and lipid hydrolysis that subsequently increase AVs. Since the presence of essential oil in mayonnaise may inhibit microbial growth due to its antimicrobial properties and subsequently reduce the production of microbial organic acids, the AVs of CEO and CEON samples may be lower compared with the control sample. The findings of the current study proved this fact and indicated that the addition of CEO and CEON to mayonnaise affected the AV.

| Effect of CEON and CEO on microbial growth
The growth of microorganisms depends on the pH, temperature, and storage time of mayonnaise (Yolmeh et al., 2014). The essential oils can more effectively inhibit microbial growth when the pH of mayonnaise is low. This is because those easily dissolve in the lipid layer of the bacterial membrane in acidic mayonnaise due to their hydrophobic properties. As a result, the destruction of the bacterial membrane leads to the leakage of cellular compounds and the inhibition of cellular activities related to the membrane, resulting in cell death (Burt, 2004;da Silva & de Melo Franco, 2012;Holley & Patel, 2005;Lambert et al., 2001;Marchese et al., 2017;Smith-Palmer et al., 2001). As shown in Figure   Although, CEON was more efficient than CEO in controlling the microbial growth and oxidative changes due to more efficiency and better performance of emulsion droplets of essential oil at the nanoscale. The evaluation of sensory attributes of mayonnaises indicated that the CEO sample obtained lower scores than CEON and control samples due to its undesirable color and odor. Based on the results, Cuminum cyminum L. essential oil nanoemulsion, due to its desirable sensorial characteristics, oxidative stability, and controlling microbial growth, can be recommended as an appropriate alternative to synthetic antioxidants and preservatives in food products.

ACK N OWLED G M ENTS
We gratefully appreciate the cooperation of all the current study participants.

FU N D I N G I N FO R M ATI O N
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Even though adequate data have been given in the form of tables and figures, all authors declare that if more data are required, then the data will be provided on a request basis.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

CO N S E NT TO PA RTI CI PATE
All the coauthors were willing to participate in this manuscript.

CO N S E NT FO R PU B LI C ATI O N
All authors are willing for the publication of this manuscript.