Industrial Peanut Oil‐Cake as a Base Material for the Construction of Nanocapsules Loaded With Oregano Essential Oil for Food Delivery Applications

ABSTRACT

The objective was to develop and characterize nanocapsules (NPs) employing a peanut oil‐cake concentrate (POCC) obtained from the industrial peanut oilcake as wall material incorporated with oregano essential oil (OEO) as core. POCC was employed to fabricate oregano essential oil nanocapsules (OEO‐NPs) with different POCC/OEO ratios: control (NPs‐0), ratio 1:1 (OEO‐NPs‐1) and ratio 1:5 (OEO‐NPs‐5). The average diameter of NPs‐0 was 404.00 ± 1.00 nm. The nanoparticle size decreased from 190.63 ± 1.00 nm for OEO‐NPs‐1 to 164.53 ± 1.00 nm for OEO‐NPs‐5. The zeta potential found for the different NPs samples highlighted that a long‐term stability of the nanoparticles can be expected. The polydispersity index (PDI) value decreased as the amount of OEO added increased. OEO‐NPs‐5 presented a higher monodispersion in comparison to the other treatments. No porosity or crack zones were detected on the surface of the NPs in the scanning electron microscopy analysis. The encapsulation efficiency (EE) was 91.33%, and 85.15% for OEO‐NPs‐1 and OEO‐NPs‐5, respectively. According to the results from the in‐vitro OEO release study, the delivery system made by POCC was suitable for controlling the release of OEO. The storage study carried out at 4°C for 90 days showed a decrease in the oxidizing capacity during storage. In general, no significant differences were observed in the volatile compounds profile for OEO‐NPs‐1 and OEO‐NPs‐5 during 90 storage days. The POCC demonstrated a good capacity to incorporate OEO with good EE and high release rate control, indicating that NPs made with POCC have potential for food preservation.

Practical Applications

Nanocapsules formed from a valueless by‐product of the peanut industry and the incorporation of oregano essential oil demonstrated an antioxidant effect, representing an alternative in the food industry for food preservation.

1. Introduction

Despite the need to increase food production in order to meet the demands of a rapidly growing population, innovative practices are required to minimize the generation of significant agricultural wastes which can release toxic products into the environment. Fortunately, circular business models actually look for strategies to convert waste into a source of rich valuable compounds. This concept is referred to as “the waste to value conception” and comprises approaches focused on convert waste materials into useful and marketable products (Phillip et al. 2024).

As one of the foremost valuable oil‐seed crops worldwide, peanuts generate a multitude of by‐products during the oil extraction process. Peanut meal, also known as peanut oilcake (POC), is a by‐product generated during the peanut oil extraction process (Lakhlifi El Idrissi et al. 2024). It consists of a brown color powder or granules rich in proteins, polypeptides, amino acids, dietary fiber, carbohydrates, lipids, vitamins, and minerals. Due to its high nutritional value, low cost and good functional properties, peanut protein has been used as source of functional ingredients in bakery, dairy, meat products, biopolymer films, stabilization of pickering emulsions, plant‐based meat analogs, and bioactive delivery systems (Cui et al. 2023; Mohamed et al. 2024; Nie et al. 2024; Suleman et al. 2023).

In recent years, Nanocapsules’ (NPs) technology has developed as one of the most encouraging scientific field of research in the food sector. In this technique, bioactive compounds are coated with a wall material to form capsules with a size of less than 1000 nm (Tahir et al. 2021). In this context, proteins appeared as a favorable wall material for the preparation of NPs because it is an amphiphilic substance with a high proportion of hydrophobic amino acids, which can produce protein nanoparticles through hydrophobic interactions (Yang et al. 2021).

Various plant‐based protein nanoparticles have demonstrated their potential as attractive carriers for encapsulation of bioactive molecules such as essential oils (EOs) (M. Zhang et al. 2022). Particularly, oregano essential oil (OEO) is considered to be a natural antioxidant and preservative, often used as a natural food additive (López et al. 2024). Regardless of their exceptional properties, EOs have a short lifetime due to their volatility and sensitivity to ambient conditions (Xu et al. 2023). The low water solubility of EOs indirectly affects their shelf life by limiting homogeneous dispersion in aqueous systems and promoting phase separation, which increases the exposure of volatile compounds to environmental factors such as oxygen, temperature, and light (Yammine et al. 2024). Thus, encapsulation of EOs could have the potential to overcome its susceptibility by enhancing their food application.

Despite advantages of nontoxicity, biodegradability, good functional properties and good thermal stability, the use of peanut protein to produce NPs is still poor (Li et al. 2015). No studies have been conducted using industrial POC as a raw material to obtained NPs. The strategy of producing high protein concentrates directly from the industrial POC would reinforce the implementation of the “value to waste” concept mentioned above in the peanut industry (Zhao et al. 2023). What is more, by means of industrial POC valorization, it is possible to generate economic opportunities for agricultural and food businesses while mitigating the environmental impacts associated with waste disposal (Phillip et al. 2024).

In view of the above, the objective of the present study was to develop and characterize NPs employing a peanut protein concentrate obtained from the industrial POC as wall material added with OEO as core material for food delivery applications.

2. Materials and Methods

2.1. Materials

Residual POC was obtained as a by‐product of the mechanical pressing of peanut seeds during oil extraction at 75°C–85°C from the company Nutrin, Córdoba, Argentina in 2022. OEO was provided by Cooperative Group called Aromaticas Alto Valle, Rio Negro, Argentina. Chemical agents used in all determinations and formulations were of analytical grade.

2.2. EO Analysis

The OEO was analyzed according to Asensio et al. (2017) with a Perkin Elmer Clarus 600 GC–MS (Shelton, Connecticut, USA) coupled with a mass detector (MS) and nonpolar capillary column Elite‐5 MS (methylpolysiloxane, 5% phenyl, 30 m, 0.25 mm id, and 0.25 mm coating thickness). The compounds were identified by comparing their mass spectra with those from libraries (NIST) and literature. In addition, the main components were identified by authentic standards (Sigma R, St. Louis, Missouri, USA). The amount of each component was calculated by peak area normalization, and the response factor was one for each component.

2.3. Preparation of Residual Peanut Oil‐Cake Flour (POCF) and Peanut Oil‐Cake Concentrates (POCC)

The methodology of Gayol et al. (2013) with slight modifications was used. POC was ground to obtain POCF which was sieved (sieve Mesh 0.75 mm). For preparing POCC, protein extraction from POCF was done. An aqueous solution was formed with a 10:1 water/flour ratio, adjusted to pH 9 with 1 M NaOH and stirred for 30 min at 40°C. The obtained slurries were centrifuged at 12,000 × g for 20 min at 25°C in a Thermo Scientific Sorvall ST 40R centrifuge (Germany). The proteins in the supernatant were precipitated at pH 4 with 1 M HCl and centrifuged at 12,000 × g for 20 min at 25°C to separate proteins and obtain a wet POCC. The obtained precipitate was re‐dispersed in distillated water. The dispersion was homogenized and adjusted to pH 7.0 with 1 M NaOH solution. The remaining water in the POCC was removed using a freeze dryer (TriEpic, Dara‐lyo, Spain). The POCC powder was stored in caramel‐colored glass jars at −4°C.

2.4. POCF and POCC Proximal Composition

Percentages of lipids, moisture, protein, and ash were determined according to AOAC (2010) methods. The Kjeldahl method was used to determine the protein percentage and the 5.46 conversion factor was used. Total carbohydrates content was estimated by difference of the other components using the following formula (Gayol et al. 2013):

$$\begin{array}{ccc}\hfill \mathrm{Carbohidrates}\mathrm{content}& =& 100\%-\left(\%\mathrm{lipids}+\%\mathrm{moisture}\right.\hfill \\ & & \left.+\%\mathrm{proteins}+\%\mathrm{ash}\right)\hfill \end{array}$$

2.5. Determination of Functional Properties of POCF and POCC

Water holding capacity (WHC) was determined following the procedure delineated by Wu et al. (2009) with slight modifications. Note that 1 g (W ₀) of POCF and POCC were placed into a 15 mL centrifuge tube, then both were weighed (W ₁). Subsequently, 10 mL distilled water was introduced into each sample and agitated using a Fisher Gene II vortex during 2 min. Then, the samples were allowed to settle at room temperature for 30 min and centrifuged at 3000 × g for 20 min. The liquid supernatant was poured off and the sediment was reweighed then (W ₂). WHC was calculated using the following equation:

$$\mathrm{WHC}=\left(W_2-W_1\right)/W_0$$

Foaming capacity (FC) and foaming stability (FS) were carried out according to de Paiva Gouvêa et al. (2023) with slight modifications. Concentrations of 1 % POCF and POCC were prepared in ultrapure water (MilliQ) and adjusted to pH 7 with 1 N NaOH. Volume of 100 mL (V₀) of each sample was blended for 3 min using an Ultra‐Turrax homogenizer (T25, Ika‐Werke, Staufen, Germany), dump into a graduated cylinder, and the volume of foam was registered at 0 min (after blending) (V ₁) for FC, while FS was measured as the percentage of foam remaining after 30 min and 60 min (V ₂), respectively according to the following equations (de Paiva Gouvêa et al. 2023):

$$\mathrm{FC}\left(\%\right)=\frac{(V_1-V_0)}{V_0}\times 100$$

$$\mathrm{FS}\left(\%\right)=\frac{V_2}{V_1}\times 100$$

2.6. Preparation of oregano essential oil nanocapsules (OEO‐NPs)

The OEO‐NPs were obtained according to the method described by Yi et al. (2015) with minor changes. First, an aqueous phase was prepared by scrambled 1 g of POCC in 100 mL ultrapure water for 24 h at room temperature. Soy lecithin was used as surfactant at a concentration of 3% w/v (relative to total emulsion volume). At the same time, an oil phase was produced by adding OEO to ethyl acetate solution. Finally, the oil phase was mixed with the aqueous phase at a ratio of 1:2. Three mixtures were prepared. The final concentration of OEO in each mixture was 0%, 1%, and 5% w/v with respect to the aqueous phase. The final relation of POCC/OEO in each mixture was 1 g of POCC and 0 g of OEO (ratio 1:0), 1 g of POCC and 1 g of OEO (ratio 1:1), and 1 g of POCC and 5 g of OEO (ratio 1:5), respectively. The different mixtures were first homogenized for 15 min at 12,000 rpm using an Ultra‐Turrax homogenizer (T25, Ika‐Werke, Staufen, Germany) and then, homogenized using a high‐pressure homogenizer (Emulsiflex Avestin C5 made in Canada) for 30 cycles at a pressure of 105 MPa. After the homogenization, ethyl acetate was evaporated from the emulsion by rotary evaporation at 28°C. The samples were then lyophilized for 24 h and kept frozen until use. Unloaded nanoparticles were synthesized and used as control. The different treatments evaluated were: NPs‐0 (unloaded nanoparticles), OEO‐NPs‐1 (nanoparticles with a POCC/OEO ratio 1:1), and OEO‐NPs‐5 (nanoparticles with a POCC/OEO ratio 1:5).

2.7. OEO‐NPs Characterization

2.7.1. Recovered Solid Yield (SY)

The recovered SY of OEO‐NPs obtained from POCC was estimated based on the following:

$$\mathrm{SY}=\left(\frac{W_f}{W_i}\right)\times 100$$

where W_f is the weight (g) of the OEO‐NPs obtained and W_i is the weight (g) of the solid constituents in the prepared emulsions (except water and ethyl acetate) (Asensio et al. 2017).

2.7.2. Particle Size, Zeta Potential, and Polydispersity Index (PDI)

The particle size, zeta‐potential and PDI of OEO‐NPs were determined by dynamic light scattering using a Malvern Zetasizer Nano‐ZS (Malvern Instruments, Worcestershire, UK) at 25°C. Refractive indices of 1.45 for OEO‐NPs and 1.33 for ultrapure water were used. The OEO‐NPs were diluted 100 times before measuring (Yi et al. 2015).

2.7.3. Scanning Electron Microscopy (SEM) Analysis

OEO‐NPs were examined using a SEM (Field Emission Gun Scanning Electron Microscope, FEG‐SEM, Carl Zeiss, Oberkochen, Alemania). The samples were prepared diluting 1 g in 20 mL ultrapure water. Then the dispersion was sonicated for 4 min at 25°C (Yang et al. 2021).

2.7.4. Encapsulation Efficiency (EE)

To explore EE, free surface OEO and total OEO contents in the OEO‐NPs were determined using a spectrophotometer following the method described by Yang et al. (2021) with minor changes. Two separate suspensions were prepared by dissolving 100 mg of freeze‐dried OEO‐NPs in 20 mL ethanol. For surface oil determination, the suspension was gently shaken at room temperature for 5 min. For total OEO quantification, a second suspension was sonicated for 60 min at room temperature. Afterward, both suspensions were centrifuged at 9000 × g and 25°C for 5 min. The resulting supernatants containing free surface oil and total oil contents, respectively, were collected for analysis at 275 nm with a DU‐730 UV/vis spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA). The absorbance was converted to OEO concentration based on the established standard curve (R ² = 0.9487). EE was estimated by the following equation:

$$\mathrm{EE}\left(\%\right)=\frac{(W_1-W_2)}{W_0}\times 100$$

where, W ₁ is the total mass (g) of oil determined spectrophotometrically after sonication of OEO‐NPs/ethanol suspension, W ₂ is the mass (g) of oil on the OEO‐NPs surface determined spectrophotometrically after shaking OEO‐NPs/ethanol suspension, and W ₀ is the mass (g) of oil added into the system during OEO‐NPs preparation. To calculate W ₀, the amount of oil required to be added to the system to produce 100 mg of nanoparticles was considered, based on the total solids composing the nanoparticles (POCC, OEO, and soy lecithin).

2.7.5. In‐Vitro OEO Release

The in‐vitro OEO release from NPs was conducted through vertical Franz diffusion cells, at 25 ± 0.5°C under continuous agitation (n = 2).The area for diffusion was 3.14 cm² and the acetate celulose dialysis membranes (10,000 Da, Sigma Aldrich) were fitted between donor and receptor compartments. The receptor compartment was filled with 17 mL of previously filtered (45 µm) and degassed phosphate buffer pH = 7.5. Dialysis membranes were previously hydrated in this medium for 24 h. The OEO‐NPs (60 mg) corresponding to 7.6 mg OEO (OEO‐NPs‐1) and 23 mg OEO (OEO‐NPs‐5) were resuspended in 1 mL of the same buffer and placed on the hydrated membrane surface. At fixed times (0, 0.25, 0,50, 1, 2, 4, 6, 24, 48 and 72 h), 1 mL of the receptor medium was withdrawn and replaced by the same volume of fresh medium at 25 ± 0.5°C (Gehrcke et al. 2021). At each time, the amount of OEO released (mg) was determined using the spectrophotometric conditions previously mentioned in Section 2.7.4 by considering the volume of phosphate buffer used as the receptor medium. Results were expressed as cumulative OEO release percentage obtained by dividing the cumulative amount of OEO released at each time point by the initial mass of OEO incorporated into the samples, according to Hosseini et al. (2013).

2.8. Antioxidant Capacity and Volatile Compounds Profile of OEO‐NPs During Storage

OEO‐NPs were stored at 4 ± 0.5°C considered as refrigerator temperature for 90 days. Antioxidant activity and volatile compounds were analyzed every 30 days.

2.8.1. Antioxidant Activity

The radical‐scavenging activity (RSA) of OEO‐NPs was analyzed using diphenyl picryl hydrazyl radical (DPPH), following the method of Xu et al. (2023) with some modifications. Aliquots of 100 µL of the solution (100 mg in 10 mL ethanol) were mixed to 900 µL of ethanolic DPPH solution. The sample was shaken and left to stand for 30 min in a dark room. Finally, the absorbance was measured at 517 nm with a spectrophotometer. To calculate the RSA, the following equation was used:

$$\begin{array}{ccc}\hfill \%\mathrm{RSA}& =& \frac{1-\left(\mathrm{absorbance}\mathrm{of}\mathrm{DPPH}\mathrm{and}\mathrm{sample}\mathrm{absorbance}\mathrm{of}\mathrm{sample}\right)}{\mathrm{absorbance}\mathrm{of}\mathrm{DPPH}}\hfill \\ & & \times 100\hfill \end{array}$$

2.8.2. Volatiles Compounds

The volatile compounds of OEO‐NPs were studied to analyze the release profile of OEO from OEO‐NPs samples during storage (Asensio et al. 2017). Samples (2 g) were weighted and added into 20 mL capacity vials and heated for 20 min at 70°C. The volatile compounds were captured by a SPME fiber of divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm, StableFlex, 1 cm long (Supelco, Bellefonte, PA, USA) in the headspace of the vial for 10 min and then, injected into the GC‐MS. Compounds were separated by a GC Perkin Elmer Clarus 600 coupled with an MS using an Elite‐5MS column (30 m × 0.25 mm i.d., 0.25 mm film thickness; Perkin Elmer). The injector temperature was 250°C. The initial temperature was maintained at 60°C for 2 min and was gradually increased to 180°C at a rate of 5°C/min and held for 2 min. Subsequently, the temperature was increased to 250°C at a rate of 5°C/min and maintained for 1 min. The components were identified with reference to the NIST mass spectral library, and the relative content of each component was calculated using the peak area normalization method.

2.9. Statistical Analysis

All experiments were carried out in triplicate. Data were analyzed using the Infostat software, version 2020p. Means and standard deviations were calculated. Analysis of variance (ANOVA) and LSD‐Fisher test were performed to determine significant differences between means (α = 0.05). Pearson´s correlation coefficients were determined for the analyzed dependent variables. Principal component analysis (PCA) was performed to assess the associations between physicochemical properties, EE, in‐vitro OEO release, antioxidant activity, and the major volatile compounds of the fresh OEO‐loaded nanoparticles.

3. Results and Discussion

3.1. EO Analysis

The chemical composition of OEO is shown in Table 1. The major compounds were γ‐terpinene (24.26 ± 0.09%), thymol (17.52 ± 0.05%), terpinen‐4‐ol (11.43 ± 0.02%), and α‐terpinene (9.04 ± 0.03%). A similar composition was found by other authors in OEO obtained from leaves and flowers (López et al. 2023; Asensio et al. 2017). Several authors have previously assigned the OEO antioxidant activity to some specific OEO compounds. For instance, Lee et al. (2015) indicated that γ‐terpinene, thymol, and terpinen‐4‐ol exert antioxidant activity in a study of EOs from Chrysanthemum indicum Linné flowers. While according to Ojeda‐Sana et al. (2013), terpenes (e.g., β‐myrcene) were the cause of the antioxidant activity of rosemary EO. Moreover, effects of OEO on oxidative stability of different foodstuffs have been also studied (Asensio et al. 2019; Benassila et al. 2025). By determining the major components of OEO, it would be expected that they can exert a protective effect against lipid oxidation.

TABLE 1.

Means and standard deviations (n = 3) of chemical compounds of oregano essential oil.

Components	Retention index	Areas relative composition (%)	Methods of identification
α‐tujene	923	1.02 ± 0.01E	GCMS
α‐pinene	933	0.99 ± 0.02E	GCMS‐Co
Camphene	952	0.02 ± 0.02A	GCMS
Sabinene	973	3.14 ± 0.01I	GCMS‐Co
β‐pinene	980	0.34 ± 0.01B	GCMS‐Co
β‐myrcene	991	1.01 ± 0.01E	GCMS
α‐phellandrene	1.005	0.49 ± 0.01C	GCMS
α‐terpinene	1.018	9.04 ± 0.03N	GCMS
p‐Cymene	1.023	6.61 ± 0.01M	GCMS
γ‐terpinene	1.059	24.26 ± 0.09Q	GCMS‐Co
Cis sabinene hydrate	1.069	0.88 ± 0.01D	GCMS
Terpinolene	1.084	3.98 ± 0.02J	GCMS
Linalool	1.098	5.53 ± 0.01K	GCMS
Camphor	1.143	1.66 ± 0.01F	GCMS
α‐terpineol	1.171	2.43 ± 0.01H	GCMS‐Co
Terpinen‐4‐ol	1177	11.43 ± 0.02O	GCMS
Thymol	1.311	17.52 ± 0.05P	GCMS
Carvacrol	1.313	6.26 ± 0.01L	GCMS‐Co
Caryophillene	1.418	1.89 ± 0.01G	GCMS
Germacrene D	1.475	0.86 ± 0.02D	GCMS
Total composition		99.39

Note: Means and standard deviations followed by different letters indicate significant differences between compounds (ANOVA, LSD test, α = 0.05).

3.2. POCF and POCC Proximal Composition

The proximate analysis of POCF and POCC is shown in Table 2. In the present study, the methodology of Gayol et al. (2013) was used to obtain POCC from POCF. Those authors emphasized the fact that a washing in the residual POCF with 70% ethanol may not be necessary since no significant increase was obtained in the final protein yield. This is important because it allow a minimum processing on the industrial POC resulting in lower costs of POCC production. In the present assay a maximum of 77.17 g/100 g of protein in POCC was obtained in comparison to 86.22 g/100 g protein obtained by Gayol et al. (2013). Past studies carried out by Wu et al. (2009) achieved peanut protein concentrates with 96.65 g/100 g in protein using the same methodology. Gayol et al. (2013) indicated that the emulsion formed during the extraction process may be affected by the fatty matter, generating a decrease in the amount of protein remaining in the concentrates. This means that the amount of proteins in the concentrate depends on to the amount of lipids in the peanut flour used. The amount of lipids in POCF in the present research was 3.41 g/100 g which could affect the protein extraction efficiency.

TABLE 2.

Proximate composition (g/100 g on wet basis) of residual peanut oil‐cake flour (POCF) and peanut oil‐cake concentrates (POCC).

Component (%)	POCF	POCC
Lipids	3.41 ± 0.06B	1.02 ± 0.03A
Proteins	43.59 ± 0.22A	77.17 ± 0.11B
Ash	4.06 ± 0.04B	2.07 ± 0.03A
Moisture	6.19 ± 0.04B	1.37 ± 0.01A
Carbohydrates	42.75 ± 0.73B	18.37 ± 0.62A

Note: Means and standard deviations followed by different letters in each row indicate significant differences (ANOVA, LSD test, α = 0.05).

According to Hariharan et al. (2023), protein isolates are those where the amount of protein is greater than 90 % and protein concentrates when the amount ranges between 68% and 72%. In the present trial, a protein concentrate was achieved.

The amount of carbohydrates ranked second in the proximal composition of POCC. Previous investigations have demonstrated that carbohydrates fraction of POCCs contain structural polysaccharides, dietary fiber, and oligosaccharides (H. Liu et al. 2016). Y. Liu et al. (2012) developed microcapsules where proteins and polysaccharides predominated, the synergy of these components allowed to obtain microcapsule walls with higher density and mechanical strength. Additionally, C. Zhang et al. (2024) indicated that polysaccharides extend the functional properties of proteins, thereby improving the stability of protein delivery systems. Probably, residual carbohydrates remained in POCC, used as wall material in the present study, interacted with peanut protein matrix and contributed to NPs stability and bioavailability through protein–polysaccharide interactions (H. Liu et al. 2016).

3.3. Determination of Functional Properties of POCF and POCC

3.3.1. WHC, FC, and FS

WHC, FC, and FS are presented in Table 3. WHC is a relevant parameter for evaluating protein–water interaction. This influences the preparation of the emulsion entering the high‐pressure homogenizer (Chen et al. 2017). The mechanisms of denaturation in the POCC preparation from POCF were heating and addition of a 1 M HCl at pH 4 and a strong base 1 M NaOH at pH 9. The strong acid and base breaks the hydrophilic and hydrophobic sites (Wu et al. 2009). These factors resulted in the decrease of WHC from POCF to POCC as stated in Table 3. This decrease in WHC could be associated with the lower carbohydrate content of POCC compared to POCF. The polymeric structure of carbohydrates enhances their ability to adsorb and retain water molecules, contributing significantly to WHC (Kelch et al. 2025). The study carried out by Wu et al. (2009) showed WHC values ranging from 1.25 g/g to 2.75 g/g for different protein concentration methods. Bühler et al. (2020) reported values ranging from 1 to 4 g/g, modifying faba bean protein concentrate by means of dry heat to increase the WHC.

TABLE 3.

Functional properties of peanut oil‐cake flour (POCF) and peanut oil‐cake concentrate (POCC).

Sample	Water holding capacity (g/g)	Foaming capacity (%)	Foaming stability (%)
			30 min	60 min
POCF	3.2 ± 0.0B	26.2 ± 0.3A	99.9 ± 0.3B	99.1 ± 0.3B
POCC	2.9 ± 0.0A	45.0 ± 0.1B	95.3 ± 0.2A	94.5 ± 0.0A

Note: Means and standard deviations followed by different letters within the same column are significantly different (ANOVA, LSD test, α = 0.05).

Foaming properties are analogous to the emulsifying capacity of a material. In foams, air acts as the dispersed nonpolar phase and water as the continuous polar phase. The amphipathic nature of proteins allows them to localize at the air–water interface, reducing surface tension and preventing bubble coalescence. In a similar way, it would behave in the formation of emulsions (Yu et al. 2007). Table 3 shows that POCF is not a good foaming agent, given its FC of 26 %, while POCC has a higher value of 45 %. This demonstrates that protein denaturation promotes amphipathicity by significantly increasing FC. Similar results were reported by Wu et al. (2009). In contrast, FC values greater than 70 % were recorded for other flours and concentrates derived from different commercial legumes, such as peas, beans, and soybeans (de Paiva Gouvêa et al. 2023). Although POCC exhibited lower FS than POCF after 30 and 60 min (Table 3), its higher initial foam volume suggested a greater potential for emulsification.

3.4. OEO‐NPs Characterization

3.4.1. Recovered SY

The recovered SY represented the weight of the powder obtained after ethyl acetate and water evaporation process through rotary evaporation and lyophilization, respectively; relative to the total weight of the components used in the emulsion formation (POCC, soy lecithin and OEO). The SY was 28.87 % for unloaded NPs (NPs‐0), while for OEO‐NPs‐1 and OEO‐NPs‐5 the yield was 30.27 % and 71.4 %, respectively. This indicates that the higher the OEO percentage added, the higher the yield. A similar study of microcapsules with OEO obtained yields ranging from 31% to 51.8% for different wall compositions (Asensio et al. 2017).

Fraj et al. (2019) observed that, even under the same processing conditions, a small increase in OEO resulted in NPs with lower EE due to an effect of oiling off. These observations, along with the higher recovered SY for OEO‐NPs‐5, suggest that a fraction of free OEO may have retained on the particle surface or physically trapped within the final dried powder after the lyophilization process. This surface oil could contribute to the final recovered powder mass, thus increasing the recovered SY of OEO‐NPs‐5. De Barros Fernandes et al. (2014) evaluated the influence of the total solid and EO concentration on the microencapsulation of rosemary EO. They highlighted that while increasing the wall material/EO ratio improves encapsulation, this efficiency has a limit. Excessive increase in the initial solids content during emulsion preparation results in lower recovered SY.

3.4.2. Particle Size, Zeta Potential, and PDI

The average size, zeta potential, and PDI of the different NPs samples are shown in Table 4. The average diameter of control samples was 404.00 nm, and the average size of the OEO‐NPs gradually decreased as more OEO was added. The nanoparticle size decreases from 190.63 nm for OEO‐NPs‐1 to 164.53 nm with increasing incorporation of OEO (OEO‐NPs‐5). Conversely, previous authors found different results. For example, a study performed by Yang et al. (2021) on preparation and characterization of cinnamon essential oil (CEO) NPs, found an increase in average size from 195.17 nm in control nanoparticles to 215.00 nm with the addition of EO. Other authors reported that chitosan NPs loaded with clove EO had a mean size of 203.47 nm and this size increased as the amount of EO increased (Xu et al. 2023). Hu et al. (2018) observed a similar result. Nevertheless, in the present study, as the amount of OEO increased, the diameter of the NPs decreased. Asensio et al. (2020) found similar results for OEO nanoemulsions and stated that the decrease in droplet size during high pressure homogenization is due to effects of viscosity and interfacial tension that occurs between the oily and aqueous phase at high pressure. Similar behaviors were observed for ginger EO containing nanoemulsions (Alexandre et al. 2016).

TABLE 4.

Particle size (Z‐average), zeta potential, and polydispersity index (PDI) of unloaded nanoparticles (NPs‐0) and oregano essential oil containing nanoparticles (OEO‐NPs‐1 and OEO‐NPs‐5).

Sample	Z‐average (nm)	PDIa	Zeta‐potencial (mV)
NPs‐0	404.00 ± 1.00C	0.449 ± 0.001B	39.0 ± 0.1A
OEO‐NPs‐1	190.63 ± 1.00B	0.305 ± 0.002B	39.6 ± 0.1B
OEO‐NPs‐5	164.53 ± 1.00A	0.266 ± 0.002A	39.0 ± 0.1A

Note: Means and standard deviations followed by different letters in each column indicate significant differences between samples (ANOVA, LSD test, α = 0.05).

The zeta potential measurement indicates molecular repulsion/attraction and interactions between particles. It is an essential parameter for analyzing the stability of a dispersion of suspension and emulsion. A highly negative or positive zeta potential (± 30 mV) indicates high repulsive forces and low probability for nanoparticles to agglomerate (Fraj et al. 2019). The zeta potential found in the current study for the different NPs samples (Table 4) highlighted that a long‐term stability of the nanoparticles can be expected. The PDI is a measure of the monodispersity of the particle size populations. The PDI value decreased as the amount of OEO added increased. These indicate that OEO‐NPs‐5 presented a higher monodispersion in comparison to the other treatments. Similar results were observed by Yang et al. 2021.

3.4.3. SEM

The SEM images in Figure 1 show the size and morphology of the NPs samples. It was observed that OEO‐NPs were relatively uniform in size, with approximately 100–200 nm and smooth surfaces. OEO‐NPs‐1 and OEO‐NPs‐5 showed a spherical shape, but NPs‐0 (control) had a square shape. The morphological differences observed between unloaded NPs and OEO‐NPs could be attributed to the effect of OEO incorporation on structural organization of the POCC matrix during NPs preparation. It has been well documented that, during nanoemulsion formation, EO droplets naturally adopt spherical morphology to reduce interfacial tension, and subsequent polymer deposition at the oil–water interface stabilizes and preserves this shape during solvent evaporation and lyophilization (Lammari et al. 2020; Mariyate and Bera 2022). This could explain the spherical morphology observed for OEO‐NPs‐1 and OEO‐NPs‐5 which is consistent with previous findings (Xu et al. 2023; Yang et al. 2021). In contrast, in the absence of OEO (NPs‐0), no hydrophobic core was formed to produce the interfacial organization, and the polymeric matrix may have undergone rearrangement or aggregation during ethyl acetate evaporation and freeze‐drying, resulting in more irregular or angular structures (square shapes). It should be noted that nanoparticle morphology is strongly influenced by several factors, including type of wall material, type of encapsulated bioactive compound, and preparation process (Yammine et al. 2024). In consequence, the morphology observed for NPs‐0 could be a combination of the behavior of the nanoemulsion formed by the wall material employed in the present study under the method used for NPs formation. For this reason, similar square shapes for other unloaded NPs are less frequently described in literature. However, Magri et al. (2025) reported irregular, rhomboidal shapes for unloaded nanoparticles made from different wall materials and spherical shapes for nanoparticles incorporated with EO.

FIGURE 1.

Scanning electron micrographs (SEM). (a) Control (NPs‐0), (b) OEO‐NPs‐1, (c) OEO‐NPs‐5.

OEO‐NPs‐5 agglomerated in clusters, but OEO‐NPs‐1 were observed independent of each other. No porosity or crack zones were detected on the surface of the NPs indicating the effective encapsulation process. Other studies performed on clove EO loaded chitosan NPs (Xu et al. 2023), chitosan NPs with CEO (Yang et al. 2021), and resveratrol nanoencapsulated (Ahmad and Gani 2021) showed homogeneous, smooth agglomerates and the size varies between 100 and 200 nm.

3.4.4. The EE

EE was used to characterize the percentage of OEO inside OEO‐NPs. Spectrophotometric analysis at a wavelength of 275 nm was performed to measure the OEO content in the OEO‐NPs. The EE was 91.33 ± 2.66% and 85.15 ± 2.08% for OEO‐NPs‐1 and OEO‐NPs‐5, respectively, with significant differences between OEO treatments (p = 0.0338). This shows an increase in EE as the percentage of OEO added decreased. Fraj et al. (2019) found similar trends in polycaprolactone NPs loaded with OEO. Those authors reported that a small increase in OEO concentration produced an effect of “oiling off” and the EE of NPs decreased. Besides, Yang et al. (2021) revealed that at low wall material concentration in the system, free CEO adheres to the surface avoiding its correct encapsulation to prepare NPs. In the present study, the lower EE in OEO‐NPs‐5 compared to OEO‐NPs‐1 might probably be due to saturation of the wall material in the former so excess of OEO was not encapsulated.

Hosseini et al. (2013) effectively encapsulated OEO in chitosan nanoparticles reporting EE from 5.45% to 24.72 % with decreasing OEO addition. Those authors used Tween 80 (T80) as a surfactant. T80 is a synthetic surfactant most commonly used as an emulsifier with good emulsification properties (Zhao et al. 2023). In the present study, it was used a natural surfactant like soy lecithin for emulsification process.

3.4.5. In‐Vitro OEO Release

Evaluation of the release behavior of NPs loaded with functional components is of outmost importance for understanding their suitability as active additives in the food industry. Solubility effects can have a great impact on how molecules are released from delivery systems such as nanoparticles (Siepmann and Siepmann 2020). The most common release mechanism of active molecules from carrier systems is diffusion, which highly depends on the size of the entrapped molecule, the medium of dispersion, and the carrier material (Yammine et al., 2024). Besides, other physicochemical release mechanisms, dependent on the carrier material characteristics, could mediate the release profile such as degradation and/or dissolution of the wall material formed by POCC and swelling due to the penetration of phosphate saline medium into the NPs. The release profile of OEO‐NPs‐1 and OEO‐NPs‐5 for 72 h is displayed in Figure 2.

FIGURE 2.

In‐vitro release profiles of OEO from nanoparticles. Values were expressed as mean ± standard deviation. Different letters indicate significant differences for the interaction treatment × time (n = 3, ANOVA, LSD test, α = 0.05).

In the present work, both OEO‐NPs exhibited a similar two‐step biphasic progression, showing an initial rapid release up to 24 h followed by a slower release of OEO up to 72 h (Figure 3). The initial rapid release up to 24 h can be attributed to the release of OEO weakly bound to the surface of NPs. Hosseini et al. (2013) found a similar biphasic process in an in vitro release study for OEO encapsulated in chitosan nanoparticles where the initial burst effect and posterior drug release rates are diminished when increasing OEO concentrations, similar to our study where a more pronounced initial burst effect was shown for NPs with a 1:1 POCC/OEO ratio (OEO‐NPs‐1) for which approximately 40% was released in the first 24 h in comparison to OEO‐NPs‐5 with 1:5 POCC/OEO ratio, which account for approximately 14% within the first 24 h.

FIGURE 3.

Biplot from the first and second components from principal component analysis (PCA). Variables: Z‐average, polydispersity index (PDI), Zeta‐potential, in‐vitro OEO release, radical scavenging activity (% RSA), encapsulation efficiency (EE), major volatile compounds (γ‐terpinene, terpinen‐4‐ol, α‐terpinene, and thymol). Treatments: OEO‐NPs‐1 and OEO‐NPs‐5. Replicates are identified by the final number in each sample description.

Regarding the second stage of OEO release, a slow release was observed from 24 to 72 h, attributed to the slow diffusion of OEO from the core of NPs to the dilution medium (Ma et al. 2022). After 48 h, there were no significant differences in the cumulative release of OEO from NPs compared to 72 h. Plateau was reached at 52.84% for OEO‐NPs‐1 and 18.29% for OEO‐NPs‐5, both corresponding to approximately 4 mg of OEO. As discussed before (Section 3.1), OEO is made up of 20 different compounds, the principals being γ‐terpinene (24.26 g/100 g) and α‐terpinene (9.04 g/100 g), both insoluble in water together with thymol (17.52 g/100 g) and terpinen‐4‐ol (11.43 g/100 g) both slightly soluble in water (approximately 1 mg/mL in water at 25°C, pH = 7.0). For instance, sink conditions are achieved for both thymol and terpinen‐4‐ol if we consider their respective percentage in OEO composition and that the substance does not exceed 20 % of its solubility in the receptor medium at any given time (Siepmann and Siepmann 2020), but not for γ‐terpinene and α‐terpinene. Assuming that water solubility of these components may not differ greatly from our phosphate saline medium, it is difficult to assume that sink conditions are attained for all OEO components, suggesting that the plateau could be reached due to a lack of solubility of OEO in the receptor medium, saturation of the acetate cellulose membrane, in addition to a diffusion control of core‐entrapped OEO to the receptor medium.

Since, for the present study, sink conditions were not achieved during the release assay, both formulations released essentially the same absolute amount of OEO (mg) at each sampling point, likely due to saturation of the membrane and/or the receptor medium. However, the cumulative release of OEO expressed in percentages and showed in Figure 2 reflected the practical differences between the two systems by normalizing the amount released relative to the initial OEO load of each formulation used. This graph shows that OEO‐NPs‐5 retained more than 80% of its encapsulated OEO available for subsequent release after 72 h, while OEO‐NPs‐1 had already released nearly 50% of its initial OEO content. Therefore, OEO‐NPs‐5 may allow a long lasting antioxidant and protective effect, as more OEO is still available to be used compared to OEO‐NPs‐1, standing out as a promising candidate to further analysis. In a real case scenario, where there is no aqueous medium and saturation will rapidly be reached, further release of OEO from NPs will require the continual removal of OEO from its acting place, which is likely to happen as OEO will continuously be acting as an antioxidant when in contact with food. Moreover, degradation, dissolution or swelling of the compact POCC nanoparticles could take place, further releasing core‐entrapped OEO. Hence, the results indicate that the delivery system made by POCC is suitable for controlling the release of OEO in saturated conditions as NPs can initially release weakly bound OEO followed by a slower release rate when saturation conditions are reached.

3.5. OEO‐NPs Storage

3.5.1. Antioxidant Activity

The results obtained from the antioxidant activity test performed on the different NPs treatments during storage are shown in Table 5. A decrease in the oxidizing capacity as the days passed was noted. The fact that phenolic compounds of EOs act as antioxidants counteracting the effects of free radicals has been widely investigated (Mahdi et al. 2021). Asensio et al. (2017) performed a storage trial for 90 days in hydroxypropylmethylcellulose and maltodextrins microcapsules incorporated with OEO stored at two different temperatures (4°C and 23°C). They found a decrease in RSA ranging from 11.81 to 3.23 over 90 days with no significant differences between temperature conditions. Those researchers reported that the higher the wall material to OEO ratio, the longer the antioxidant activity of microcapsules during storage. Similar results were found in the present work where OEO‐NPs‐5 with a lower POCC/OEO ratio (1:5) preserved in a lesser way the antioxidant capacity in comparison to OEO‐NPs‐1 (POCC/OEO, 1:1) at the end of storage period. Other investigations (Magri et al. 2025) did not report a relation between wall material/EO and antioxidant capacity. Additionally, previous investigations (González‐Reza et al. 2020) reported that the antioxidant capacity of EO‐loaded NPs is directly associated with the active compounds that effectively remained within the nanostructured system. Considering that EE of OEO‐NPs‐5 was lower in comparison to OEO‐NPs‐1 (Section 3.4.4), a greater amount of superficially adsorbed or nonencapsulated oil in the former may have undergone oxidative deterioration during the storage study, thereby reducing the antioxidant activity of OEO‐NPs‐5 in comparison to OEO‐NPs‐1 at the end of the 90‐day storage period.

TABLE 5.

Radical scavenging activity (RSA %) determined on OEO‐NPs‐1 and OEO‐NPs‐5 during 90 days of storage at 4 ± 0.5°C.

Days	RSA (%)
	OEO‐NPs‐1	OEO‐NPs‐5
0	13.43 ± 0.79D	11.07 ± 0.90C
30	12.77 ± 0.22D	10.5 ± 0.53BC
60	12.30 ± 0.22D	9.79 ± 0.19B
90	9.73 ± 1.39B	8.57 ± 0.74A

Note: Means and standard deviations followed by different letters indicate significant differences for the interaction treatment × storage days (ANOVA, LSD test, α = 0.05).

3.5.2. Volatile Release Analysis

Although the release behavior of volatile compounds from NPs containing OEO during storage is highly significant, it has not been extensively investigated. Monitoring the volatile profile of OEO‐NPs provides important information regarding nanoparticle stability, retention efficiency, controlled release behavior, and the potential effectiveness of the system for future food preservation applications. The results of the chemical composition of volatile compounds released from OEO‐NPs during 90 days of storage are shown in Table 6, where only volatile compounds with relative percentages greater than 4 % are shown. Components in relative percentages ≤ 4 % are listed in Table S1. The total components found in OEO (Section 3.1) appeared in OEO‐NPs maintaining the four major volatile compounds (concentrations higher than 4 %) detected in pure OEO (α‐terpinene, γ‐terpinene, terpinen‐4‐ol, and thymol). In general, no remarkable differences were observed in the volatile profile for OEO‐NPs‐1 and OEO‐NPs‐5 during storage. In contrast, Yang et al. (2021) reported increases or decreases in some components in CEO NPs respect to the nonencapsulated oil. Those results were attributed to the selective embedding of different CEO components in NPs. Taking that into account, nanoencapsulation process conducted here allowed an appropriate encapsulation of the different components of OEO. Asensio et al. (2019) detected an increase in volatile compounds of OEO nanoemulsions during 14 storage days highlighting the possible migration from the oil droplets into the water phase being volatilized or oxidized to unstable forms. The same result was obtained for rosemary EO microcapsules by De Barros Fernandes et al. (2014).

TABLE 6.

Volatile compounds (area relative composition, %) determined on OEO nanocapsules during 90‐day storage period at 4°C.

Volatile compounds	OEO NPs‐1				OEO NPs‐5
	Day 0	Day 30	Day 60	Day 90	Day 0	Day 30	Day 60	Day 90
α‐terpinene	7.37 ± 0.01D	7.21 ± 0.01A	7.29 ± 0.01B	7.33 ± 0.03C	8.24 ± 0.02F	8.13 ± 0.02E	8.24 ± 0.01F	8.22 ± 0.03F
p‐Cymene	5.29 ± 0.02A	5.44 ± 0.01B	5.42 ± 0.01C	5.41 ± 0.01B	5.50 ± 0.02D	5.51 ± 0.01D	5.52 ± 0.01D	5.5 ± 0.01D
γ‐terpinene	14.45 ± 0.03E	14.31 ± 0.01D	14.26 ± 0.02D	14.6 ± 0.01F	12.01 ± 0.03A	12.13 ± 0.01B	12.24 ± 0.02C	12.00 ± 0.01B
Linalool	4.54 ± 0.01A	4.52 ± 0.01A	4.53 ± 0.01A	4.54 ± 0.01A	4.63 ± 0.01B	4.64 ± 0.01B	4.63 ± 0.01B	4.63 ± 0.02B
Terpinen‐4‐ol	5.41 ± 0.01B	5.23 ± 0.01B	5.21 ± 0.01B	5.26 ± 0.01B	4.6 ± 0.01A	4.61 ± 0.02A	4.5 ± 0.01A	4.55 ± 0.01A
Thymol	46.72 ± 0.03DE	47.15 ± 0.01F	47.12 ± 0.03F	46.91 ± 0.01E	46.41 ± 0.02CD	46.38 ± 0.01AB	46.25 ± 0.01A	46.52 ± 0.02BC
Other compounds a	12.03 ± 0.02B	12.03 ± 0.02B	12.05 ± 0.01B	11.87 ± 0.01A	14.06 ± 0.02C	14.07 ± 0.01CD	14.08 ± 0.02CD	14.09 ± 0.01E

Note: Means and standard deviations followed by different letters, indicate significant differences for the interaction treatment × storage days (ANOVA, LSD test, α = 0.05).

^a Other compounds: sum of compounds in percentages ≤ 4 %.

3.5.3. PCA

Figure 3 shows a PCA plot of the different variable measurements for each OEO loaded nanoparticle (OEO‐NPs‐1 and OEO‐NPs‐5). PC1 was the most representative component, accounting for 94.4 % of the data variability, whereas PC2 was considerably low (5.4 %). A very clear distinction was observed between OEO‐NPs‐1 and OEO‐NPs‐5 samples along PC1 indicating that the concentration of EO added influences the measured properties. Furthermore, the close grouping of the replicates for each treatment indicated good experimental reproducibility. OEO‐NPs‐1 samples were located on the right side of PC1 and were linked with higher values of EE; % RSA; Zeta‐potential; PDI; Z‐average; in‐vitro OEO release; and the volatile components of OEO, such as γ‐terpinene, terpinene‐4‐ol, and thymol. Most of these variables also showed strong positive associations with each other, specifically % RSA, Zeta‐potential, PDI, Z‐average, in‐vitro OEO release, γ‐terpinene, terpinene‐4‐ol, and thymol. Asensio et al. (2019) indicated that thymol is the characteristic volatile component of the oregano mainly responsible for the antioxidant activity exhibited by its EO, which is consistent with the results of the present study, in which % RSA and thymol showed a strong positive association. On the contrary, OEO‐NPs‐5 samples were closely situated on the left side of the biplot showing association with higher α‐terpinene values. These samples were opposite to the variables mentioned above. Particularly, this treatment showed lower values for the variables PDI, Zeta‐potential, and Z‐Average as can be seen in Figure 3. As discussed in Section 3.4.2, previous authors (Alexandre et al. 2016; Asensio et al. 2020) indicated that increasing OEO concentration under high pressure homogenization may promote droplet disruption effects resulting in smaller NPs. These findings are consistent with the present study, in which increasing EO concentration at a constant wall material proportion led to smaller nanoparticles for OEO‐NPs‐5. Other authors (Yammine et al. 2024) stated that smaller nanoparticles exhibit a higher surface to volume ratio which decreases the attractive forces between droplets and promotes a better distribution and stability of the nanoemulsion, thereby lowering PDI.

The associations shown in PCA biplot were confirmed by Pearson's correlation coefficients. Correlation coefficients above 0.85 were found between % RSA and the volatiles γ‐terpinene (p = 0.0232), terpinene‐4‐ol (p = 0.0232), and thymol (p = 0.0093), which confirms the close relationship between antioxidant activity and the profile of volatile compounds of OEO‐loaded NPs. Furthermore, EE was positively linked to % RSA (R = 0.98, p = 0.0007) and thymol content (R = 0.84, p = 0.0363). This indicates that OEO‐NPs‐1, which exhibited higher EE, tended to retain greater amounts of thymol. Since this volatile compound is one of the main compounds responsible for the antioxidant activity of OEO (Asensio et al. 2019), its higher retention may have contributed to the higher % RSA observed in these samples. Furthermore, these results are consistent with those reported by González‐Reza et al. (2020) who stated better preservation of antioxidant activity in NPs exhibiting higher EE (as stated in Section 3.5.1). However, the in‐vitro release study (Section 3.4.5) indicated that OEO‐NPs‐5 retained a considerably higher proportion of its encapsulated OEO after 72 h. Therefore, despite having a lower % RSA, OEO‐NPs‐5 could provide a more prolonged antioxidant effect due to its slower release rate and its larger reserve of encapsulated bioactive compounds available for subsequent release. In addition, strong positive correlations over 0.95 were noted between Z‐average and the variables Zeta‐potential (p = 0.0022) and PDI (p = 0.0001). Accordingly, previous authors also reported a high positive correlation between PDI, Z‐average, and Zeta‐potential (Fraj et al. 2019). On the contrary, α‐terpinene showed significant negative correlations with % RSA (R = −0.85, p = 0.0329), thymol (R = −0.99, p = 0.0002), and in‐vitro OEO release (R = −1.00, p < 0.0001).

The associations and correlations revealed by PCA and Pearson´s analysis were consistent with the results obtained during NPs characterization, and the antioxidant and volatile profile study. These findings reinforce the potential of POCC as an effective wall material for producing stable OEO NPs capable of preserving antioxidant and volatile compounds while promoting a controlled EO release.

4. Conclusion

In the present study, a peanut concentrate obtained from an industrial by‐product generated by the peanut oil production like POC constitutes a good wall material to nanoencapsulate functional compounds like OEO. The POCC has suitable characteristics to embed OEO with high EE to control its release. Considering their potential preservative effect, the OEO‐NPs developed in this study may be an interesting ingredient to be used in food preservation strategies added to edible coatings or biodegradable packaging. However, further studies are needed to evaluate the preservative effects of OEO‐NPs in different matrices.

Conflicts of Interest

None.

Supporting information

Supplementary Table S1 Relative composition (%) of minor volatile compounds (≤ 4 %) determined on OEO NPs during 90‐day storage period at 4°C.