Improvement in the Stability of Perilla Seed Oil Microemulsion and Its Role in Fat Accumulation Reduction in Caenorhabditis elegans

Abstract

Perilla seed oil (PSO) possesses various physiological functions, such as lowering blood lipids and preventing cancer; however, its poor water solubility, dispersibility, and oxidative stability severely limit its application scope. Epigallocatechin gallate (EGCG) is a natural antioxidant abundant in tea leaves. In this study, PSO–casein–EGCG microemulsions were prepared, and their stability and lipid-lowering effects were evaluated. The results showed that the PSO microemulsion had a particle size of 361.23 ± 14.85 nm, a zeta potential of −20.77 ± 0.68 mV, a polydispersity index (PDI) of 0.17 ± 0.07, and an encapsulation efficiency of 94.3%. PSO microemulsions remained stable at room temperature for 5 days without droplet aggregation. The stability of the microemulsions was good when the NaCl concentration was between 0.1 and 1 mM and the pH was between 5 and 9. PSO microemulsions enhanced the oxidative stability of PSO. Additionally, PSO microemulsions significantly reduced triglyceride levels in Caenorhabditis elegans (77.50%, p < 0.005). Finally, it was found that the average lipid droplet size of ZXW618 mutant nematodes decreased by 41.23% after PSO microemulsion treatment. Therefore, PSO microemulsions may reduce fat accumulation in C. elegans by decreasing lipid droplet size. This provides new insights for advancing the application of PSO in the food processing industry.

1. Introduction

Hyperlipidemia has become a global health threat. Clinical lipid-lowering drugs mainly include fibrates, cholesterol biosynthesis inhibitors, niacin, bile acid mixtures, cholesterol absorption inhibitors, and sterol-binding protein ligands, which may cause side effects such as nausea and diarrhea [1,2,3]. Therefore, the development of functional foods with lipid-lowering effects has become a focus of attention.

Perilla seed oil (PSO) is obtained from the mature seeds of Perilla frutescens, a medicinal plant in the labiaceae family [4,5]. Generally, the contents of unsaturated fatty acids and polyunsaturated fatty acids in PSO are 91.1% to 93.8% and 70.7% to 83.4%, respectively. Alpha-linolenic acid accounts for the highest proportion, ranging from 58.8% to 70.9% [6]. PSO has various physiological benefits, such as preventing cancer, cardiovascular disease, inflammation, and even depression [7,8]. Although PSO has potential application value, its inherent characteristics—easily oxidized, poor water solubility, and low bioavailability—pose significant challenges to fully realizing its benefits [9]. In recent years, food-grade microemulsions, a form of fat in food, have been widely used in the encapsulation and delivery of functional fats [10]. The encapsulation of fat-soluble nutrients and flavor substances can prevent the oxidation of functional nutrients, improve their water solubility, and increase their bioavailability. In an oil-in-water (O/W) system, the oil phase is dispersed in the water phase in the form of tiny droplets. Its stability depends on the adsorption and assembly of emulsifiers at the oil–water interface. In this study, an O/W system was constructed to improve the water solubility and dispersibility of PSO, while the addition of EGCG enhanced the compactness of the interfacial film through interaction with casein, thereby improving the physical and oxidative stability of the microemulsion.

C. elegans is a widely used genetic model in contemporary biological analysis. Due to its short life cycle, prolific reproduction, simple anatomical structure, and ease of maintenance, it is an ideal in vivo evaluation model [11]. C. elegans has a simple, yet complete nervous system and shares a high degree of homology with human genes. Various lipid synthesis and catalytic enzymes are present in the epidermal and intestinal epithelial cells of nematodes [12]. Nematodes primarily store fat in subcutaneous and intestinal cells. Fat can be stained by binding with lipid-affinity dyes such as Nile red and ORO, and the amount of fat in nematodes can be quantified by measuring the intensity of dye accumulation in their transparent bodies [13,14]. Therefore, it is widely used as a model organism in lipid metabolism research. This study used C. elegans as the experimental subject to evaluate the lipid-lowering properties of PSO microemulsions.

In summary, this study utilized a low-temperature continuous phase-change extraction method developed by the research team to extract PSO. Compared to traditional methods, this approach achieves higher extraction rates at lower temperatures, thereby minimizing nutrient loss in the raw material. PSO–casein–EGCG microemulsions were prepared using orthogonal optimization and high-pressure homogenization, physical properties (particle size, zeta potential, pH/ionic stability) and oxidative stability of the microemulsions were characterized, and triglyceride content and lipid droplet size were measured to evaluate the lipid-lowering effect of the microemulsion on C. elegans. This study aimed to address the application limitations of PSO and provide a theoretical basis for its development as a functional food and its ingredients.

2. Materials and Methods

2.1. Materials and Reagents

Casein, PSO, and epigallocatechin gallate (EGCG) were all food-grade. NaCl, CaCl₂, MgSO₄, NaOH, K₂HPO₄, KH₂PO₄, Na₂HPO₄, NaOCl, trichloroacetic acid (TCA), thiobarbituric acid (TBA), HCl, butyl hydroxy anisol (BHA), LB broth, PBS solution, anhydrous ethanol, technical AGAR powder, tryptone, streptomycin sulfate, cholesterol, and sodium azide as domestic-level analysis reagent and a total protein quantitative test box and triglyceride (TG) test box were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China).

All experiments were conducted in accordance with the standard protocols established by the Caenorhabditis Genetics Center (CGC). As an invertebrate organism with a simple nervous system, C. elegans is not subject to formal ethical regulations and is widely exempt from institutional ethical approval requirements. The wild-type N2 strain of C. elegans was sourced from the C. elegans Genetics Center (University of Minnesota, Minneapolis, MN, USA). The ZXW618 strain [hkdIs618 (dhs-3p::dhs-3::GFP)] was provided by Zhengxing Wu’s laboratory (Huazhong University of Science and Technology, Wuhan, China). To culture these eggs, we used nematode growth medium (NGM) agar plates maintained at 20 °C, with live Escherichia coli OP50 (E. coli OP50) serving as the nutritional source.

2.2. Microemulsion Preparation

PSO was prepared using a method similar to that described in a previous report [15]. In a nutshell, PSO was extracted by a continuous-phase transition extraction under low temperature method developed by our lab, and the parameters were as follows. Raw material was treated with a 40-mesh sieve. The extraction process was carried out at a temperature of 35 °C and extraction pressure of 22 MPa for a period of 2 min. The raw material used had a particle size of 40 mesh, and the ultimate outcome of the PSO resulted in a yield of 39.7%. The oil-in-water microemulsion containing PSO was prepared by formulating an aqueous emulsifier solution with 0.5 wt% protein, specifically casein, which was stirred for a minimum of 2 h to ensure complete dissolution. The oil phase was then prepared by adding 0.01 wt% EGCG to the oil and subjecting it to magnetic stirring for 10 min, followed by ultrasonic stirring for 20 min. Subsequently, the crude microemulsion consisting of 10 wt% oil phase and 90 wt% aqueous emulsifier solution was vigorously stirred at ambient temperature for an additional 2 min. The microemulsion was prepared through five passes at 500 bars using a high-pressure homogenizer. After the preparation, all microemulsions were stored at 4 °C.

The three formula variables selected in the optimization study are shown in

Table 1. Experimental factor-level table.

Level	Factors
	A (EGCG wt%)	B (CA wt%)	C (PSO wt%)
1	0.005	0.25	10
2	0.010	0.50	20
3	0.015	0.75	30

. A standard L9 orthogonal array was used to choose the optimum microemulsion formula (

Table 2. Factor level in L9 orthogonal design experiments.

Test	A (EGCG mg/100 mL)	B (CA g/100 mL)	C (PSO g/100 mL)
1	1	1	1
2	1	2	2
3	1	3	3
4	2	2	3
5	2	3	1
6	2	1	2
7	3	3	2
8	3	1	3
9	3	2	1

). Factors were at three levels, and all experiments were repeated. Zeta potential and MDA were evaluated.

2.3. Fluorescence Spectroscopic Analysis

The casein solution was prepared at 0.5 mg/mL, while the EGCG concentrations were 0, 0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL. The excitation wavelength λ_ex = 280 nm, with an excitation slit width of 2.5 nm and an emission slit width of 5.0 nm. The fluorescence scanning speed employed was set at a rate of 1200 nm/min, covering a scanning range from 285 to 500 nm.

2.4. Microemulsion Morphology Observed by Laser Confocal Microscopy

The microstructure of microemulsions was observed using a confocal scanning laser microscope equipped with a 60× oil immersion objective (Nikon d-eclipse C1 80i, Nikon, Melville, NY, USA). In preparation for this microscopic examination, the oil droplets within the microemulsions were stained using a Nile red solution, which was prepared at a concentration of 1 mg/mL in ethanol. The excitation wavelength and emission wavelength of Nile red were set to 543 nm and 605 nm, respectively. The pinhole diameter was set to 30 microns.

2.5. Measurement of Zeta Potential

The zeta potential of microemulsion droplets was measured using a zeta potential analyzer (Zatasizer Nano ZS 90, Malvern Panalytical, Malvern, UK). The measuring chamber of the particle electrometer was filled with a diluted microemulsion and the direction was determined to calculate the zeta potential.

2.6. Determination of Droplet Size and Polydispersity Index

The average size of particles and the polydispersity index were determined by dynamic laser scattering (DLS). The microemulsion were analyzed using a nanoparticle size zeta potential analyzer. To determine the average particle size, the samples were loaded into a colorimetric dish with a refractive index of 1.33. The temperature was maintained at 25 °C for a duration of 3 min, and each experiment consisted of three independent biological replicates.

2.7. PSO Encapsulation and Loading Efficiencies

The content of free PSO was determined by visible spectrophotometry, followed by calculations to determine encapsulation efficiency and load content. Prior to preparation, 0.3% oil red O (ORO) was dissolved into the sample as a marker for quantifying the amount of free PSO through UV-visible spectrophotometry. The absorbance of ORO was detected at a wavelength of 519 nm, which is a substitute for the amount of free PSO. The PSO content in the sample was calculated using a standard curve prepared from a mixture of known PSO and ORO in hexane. The sample (10 mL) was dissolved in 20 mL hexane. The suspension was then mixed and shaken in an oscillating incubator at 250 rpm for 5 min to extract the free PSO. The obtained suspension was centrifuged at 3000 rpm for 10 min and the supernatant was collected for analysis.

2.8. Microemulsion Rheology

Steady shear viscosities of microemulsions of various pH values and ionic strength were characterized using an MCR502 rheometer (MCR502, Anton Paar, Graz, Styria, Austria) combined with a rheological cup (cp25). Apparent viscosity was measured within a shear rate range 0.1–300 s⁻¹, and a waiting process of 60 s was applied before each test. All experiments were conducted at a temperature of 25 °C.

2.9. Physical Stability of Microemulsions Under Environmental Stresses

To investigate the impact of various environmental factors on the stability of the microemulsion, the particle size, zeta potential, PDI, and rheological properties of the sample were measured at different pH levels ranging from 3 to 9 and varying concentrations of NaCl from 0.1 mM to 1 M.

2.9.1. Microemulsion pH Stability

The pH range of each microemulsion sample was adjusted using either 1.0 M or 0.5 M NaOH, and/or CH₃COOH, spanning from 3 to 9. Subsequently, the microemulsion samples were transferred into clean glass tubes and stored at a temperature of 25 °C. After a period of one day, the particle size, zeta potential, PDI, and rheological properties of the samples were measured in triplicate.

2.9.2. Ionic Stability of Microemulsions

Microemulsion samples were added to an equal volume of NaCl solutions at various concentrations ranging from 0.1 mM to 1 M. Subsequently, samples were thoroughly mixed using a vortex mixer, transferred into a clean glass test tube, and stored at a temperature of 25 °C. After one day, the particle size, zeta potential, PDI, and rheological properties of samples were measured in triplicate.

2.10. Microemulsion Oxidation Stability

Measurement of lipid peroxidation is an important indicator of microemulsion stability. Of the products produced in this process, MDA has been identified as the most mutagenic compound produced by lipid peroxidation and has been widely used for many years as a convenient biomarker for assessing the response to peroxidation of ω-3 and ω-6 fatty acids. Microemulsion samples were dispensed into sterile glass tubes and stored at 4 °C to assess sample stability. The concentration of MDA in the microemulsions was obtained by determining the thiobarbituric acid conjugate (TBARS) content of the microemulsion. The TBARS (thiobarbituric acid conjugate) level of each sample was measured after 1, 3, and 5 days. All measurements were taken in triplicate.

The TBARS measurement method was based on Caprioli with appropriate modifications [16]. A TBA solution was prepared by mixing 0.375 g of TBA, 15 g of TCA, 1.76 mL of 12 M HCl, and 82.9 mL of H₂O. Subsequently, a mixture containing 2.5 mL of this solution and 0.5 mL of microemulsion was heated in a boiling water bath for 30 min, cooled to room temperature, and centrifuged at 8000 rpm for 10 min, and the absorbance of the supernatant was measured at 532 nm. A standard curve was constructed using 1,1,3, 3-tetraethoxypropane.

2.11. Detection of Triglyceride Content in Nematodes

TG content and BCA protein assays were performed using a commercially available kit in accordance with the manufacturer’s instructions (Nanjing Jiancheng Bioengineering institute, Nanjing, China). Absorbance measurements were obtained using a Multimode Plate Reader (Perkin-Elmer, Waltham, MA, USA). All calculated TG concentrations were normalized to protein concentrations. Each experimental group consisted of at least 1000 worms aged 60 h, and the experiments were independently replicated three times.

2.12. Staining of C. elegans Using Oil Red O

Measurement of the accumulation of adipose tissue was conducted in accordance with the modified protocol [17]. To prepare the oil red O (ORO) staining solution, a concentrated stock was first created by dissolving ORO in isopropanol at a concentration of 6 mg/mL. This mixture was then left to incubate on a shaker overnight. For staining purposes, this concentrated ORO solution was further diluted with deionized water at a ratio of 60% original ORO solution to 40% deionized water. The diluted staining solution was then filtered for use. In the preparation of C. elegans for staining, the worms were initially anesthetized using a 1% solution of sodium azide. This step was followed by fixing the worms with 4% paraformaldehyde to immobilize them. The immobilized worms were then subjected to a rapid freezing process at −80 °C. To enhance the staining process, the worms underwent three thawing cycles in regular tap water (with the final cycle being frozen on ice). This procedure was designed to ensure optimal penetration of the ORO stain into the worm tissues. After paraformaldehyde removal, the specimens were dehydrated with 60% isopropanol for a duration of 15 min. Subsequently, the fixed nematodes were subjected to ORO staining solution for a period of 12 h under light-protected conditions at room temperature to ensure that all nematodes were uniformly colored. At the end of the staining procedure, visualization of the nematode was performed. Visual photography of the posterior pharyngeal intestine was performed using a CX-41 microscope (Olympus Co., Tokyo, Japan) equipped with a 40-fold objective lens. ImageJ 1.49 software was utilized to measure the intensity levels. Each experiment included more than 15 worms and was repeated three times.

2.13. Quantitative Analysis of Lipid Droplets in C. elegans Mutant ZXW618

The quantification of lipid droplets in ZXW618 worms was performed using label-free methods as previously described [18]. All worms were immobilized in a droplet of 1% sodium azide and mounted on slides prior to confocal laser scanning microscopy imaging. Fluorescent images were acquired with a 488 nm excitation filter and a 525 nm emission filter. Each experimental condition involved the analysis of over 20 worms, and the images obtained were subjected to processing using ImageJ software. The experiments were independently replicated three times.

2.14. Statistical Analysis

Each experiment was replicated in triplicate, and the data are expressed as means ± standard error. SPSS 23.0 (SPSS, Inc. [now a subsidiary of IBM Corporation], Chicago, IL, USA) was employed for data analysis. In addition, one-way analysis of variance (ANOVA) was conducted for multiple-group comparisons, and statistical significance was considered when p < 0.05. Then, the data were plotted by GraphPad Prism 9.5 software.

3. Results and Discussion

3.1. Composition Analysis of PSO and Preparation of PSO Microemulsion

In this study, PSO was extracted by a continuous phase-transition extraction method developed by our team, which carried out continuous countercurrent extraction of materials at a lower temperature, with the advantage of less solvent residue and without damaging the heat-sensitive components [17]. Subsequently, gas chromatography–mass spectrometry (GC-MS) (TSQ 8000, Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the composition. Omega-3 fatty acids were the main constituents of PSO, with α-linolenic acid accounting for 63.51% ± 5.84% of the total fatty acids (see

Table 3. Fatty acid content analysis of perilla seed oil.

Fatty Acid Composition	Palmitic Acid	Oleic Acid	Linolenic Acid	α-Linolenic Acid
Content	6.39% ± 0.78%	20.22% ± 6.64%	10.25% ± 1.23%	63.51% ± 5.84%

). In addition, other fatty acid contents in PSO were similar to other reports [19]. However, the application of PSO is notably constrained due to its limited water solubility, dispersibility, and susceptibility to oxidation. These drawbacks necessitate improvements in its stability to broaden its utility. In order to enhance the stability of PSO, an orthogonal experiment with three factors and three levels was conducted to choose the optimum microemulsion formula, including the effects of EGCG content (A), casein content (B), and PSO content (C). As shown in

Table 4. Experimental results of L9 orthogonal array design matrix and microemulsion preparation.

Test	A (EGCG wt%)	B (CA wt%)	C (PSO wt%)	MDA Increased Rate	Zeta Potential
1	1	1	1	0.12	0.97
2	1	2	2	0.27	0.70
3	1	3	3	0.16	0.62
4	2	2	3	0.27	0.93
5	2	3	1	0.25	1.00
6	2	1	2	0.39	0.96
7	3	3	2	1.00	0.77
8	3	1	3	0.07	1.00
9	3	2	1	0.40	0.94
K1	0.55	0.58	0.77	K’1	2.29	2.93	2.90
K2	0.91	0.95	1.67	K’2	2.89	2.57	2.43
K3	1.47	1.42	0.51	K’3	2.71	2.39	2.55
k1	0.18	0.19	0.26	k’1	0.76	0.98	0.97
k2	0.30	0.32	0.56	k’2	0.96	0.86	0.81
k3	0.49	0.47	0.17	k’3	0.90	0.80	0.85
R	0.31	0.28	0.30	R’	0.20	0.18	0.16

Note: K1–3, k1–3, R are the analysis results of the rate of MDA increase, and K’1–3, k’1–3, R’ are the analysis results of the zeta potential.

, when analyzing the rate of MDA increase, the order of influencing factors was: EGCG content > PSO content > casein content. When analyzing the zeta potential, the order of influencing factors was: EGCG content > casein content > PSO content. To balance oxidative stability (MDA) and physical stability (zeta potential), the optimal formulation was determined by integrating the results of both indicators.

For factor A (EGCG), A2 (0.01 wt%) was selected because it has a moderate rate of MDA increase and the highest zeta potential, and its physical stability is superior to that of A1. For factor B (casein), B2 (0.5 wt%) was selected, because although B1 has a lower rate of MDA increase, B2 balances the two indicators and has been proven to form a stable interfacial film. For factor C (perilla seed oil), C1 (10 wt%) was selected because it has a lower rate of MDA increase and stable zeta potential, thereby avoiding the aggregation risk associated with high perilla seed oil loading.

In summary, the optimal conditions were: PSO (10 wt%), casein (0.5 wt%), and EGCG (0.01 wt%). Microemulsions were prepared and characterized using the optimal formulation, with a particle size of 361.23 ± 14.85 nm, zeta potential of −20.77 ± 0.68 mV, and polydispersity index (PDI) of 0.17 ± 0.07. These characterization results indicated that the PSO microemulsion exhibited stable properties.

3.2. Microstructure of PSO Microemulsion

To understand the microstructure among PSO, EGCG, and casein, the binding condition between casein and EGCG was investigated firstly. For casein, the endogenous fluorescence spectra of proteins were determined by tryptophan, tyrosine, and phenylalanine residues. Of the three amino acids, tryptophan is the most sensitive to changes in environmental polarity and molecular structure. Therefore, it is often used as a fluorescent probe to study protein–polyphenol interaction. For EGCG, the three hydroxyl groups in the benzene ring are attached to the ester groups, which tend to form hydrogen-binding proteins and protein complexes [20]. As such, the interaction between casein and EGCG will result in fluorescence quenching. Therefore, the binding condition between casein and EGCG was determined by observing fluorescence quenching. As shown in Figure 1a, the fluorescence intensity was the highest when EGCG was not added and decreased as EGCG was added. This result suggested that EGCG bound with casein.

To further test whether PSO was bound to the EGCG–casein complex, the microstructure of microemulsions was studied. As shown in Figure 1b, the oil phase of the microemulsion dyed with Nile red showed red fluorescence at 510 nm, and the EGCG–casein complex showed green fluorescence at 325 nm. The results indicated that the oil phase in the microemulsion droplets was spherical, and the EGCG–casein complex was continuously and completely covered on the surface of the droplets. This indicated that the prepared microemulsion was spherical in shape and the EGCG–casein complex was completely adsorbed on the interface of the microemulsion. Therefore, EGCG only impacted the size of microemulsion droplets, but did not affect the micromorphology of microemulsion droplets.

3.3. Physical and Chemical Stability of PSO Microemulsions

Protein microemulsion delivery systems are widely used in complex food systems and are often exposed to harsh environmental conditions such as high salt concentrations and unstable pH values, which accelerate the oxidative deterioration of microemulsions. Therefore, characterizing the physical and chemical stability of microemulsions under different conditions is of critical importance. In this study, the physical stability of microemulsions was characterized by measuring their zeta potential, average droplet diameter, polydispersity index (PDI), and viscosity at different pH values or salt concentrations. Chemical stability was characterized by quantitatively analyzing MDA concentrations.

3.3.1. Effect of pH on Physical Stability of PSO Microemulsions

We studied the stability of PSO microemulsions at different pH values. As shown in Figure 2, the results indicated that the droplet size of PSO–casein and PSO–casein–EGCG microemulsions remained relatively stable at pH values between 4 and 9. PDI, an indicator of microemulsion uniformity, showed that the PDI value of the PSO–casein–EGCG microemulsion was lower than that of the PSO–casein microemulsion at pH values ranging from 4.0 to 8.0, indicating that EGCG enhances the uniformity of microemulsion droplets [21]. The zeta potential is an indicator of the electrostatic repulsive force between droplets. At pH values ranging from 5.0 to 9.0, the PSO–casein–EGCG microemulsion exhibited a higher net negative charge, indicating relative stability, with the net negative charge increasing as pH increases. Above the isoelectric point of casein (pH 4.6), carboxyl groups carry a negative charge, resulting in net negative charges between droplets. In the PSO–casein–EGCG microemulsion, casein, an amphiphilic phosphoprotein, spontaneously anchored at the oil–water interface due to its flexible structure, with hydrophobic domains (e.g., proline-rich regions) embedding into the PSO phase and hydrophilic groups (e.g., phosphate moieties and polar amino acids) extending into the aqueous phase. This arrangement forms a sterically hindered interfacial film that physically prevents droplet coalescence, while the disordered structure of casein enhances its interfacial adaptability, ensuring dense packing and long-term stability [22]. EGCG further reinforces this interfacial layer through hydrogen bonding with polar residues (e.g., serine, threonine) of casein and hydrophobic interactions with nonpolar regions (e.g., aliphatic amino acids), thereby tightening the interfacial network and increasing its mechanical strength. Together, these dual interactions not only improve physical stability but also contribute to oxidative resistance by scavenging free radicals at the interface [20].

As shown in Figure 3, the shear stress curves of the microemulsions at different pH values (3.0–9.0) and shear rates (0–300 s⁻¹) were measured. At pH 5.0–8.0, the shear stress was lowest. This indicated that at pH 5.0–8.0, droplet size distribution was smaller and more uniform. Under the shear rate of 0–300 s⁻¹, all the microemulsions showed shear thickening behavior and the shear stress increased with the increase in the shear rate. Shear thickening is caused by locally concentrated particles overcoming interparticle forces [10]. The droplet interaction is enhanced to make the microemulsion more stable and prevent flocculation [10]. This result showed that the combination of EGCG and protein made the microemulsions more uniform and the microemulsion droplets interacted with each other, reducing the aggregation. Therefore, combining the results of zeta potential, average droplet size, PDI, and viscosity, PSO–casein–EGCG microemulsions are stable within the range of pH 5.0–8.0.

3.3.2. Effect of Ionic Strength on Physical Stability of PSO Microemulsions

The preparation of emulsified food varies greatly with the ionic strength of the product. In general, ion concentration is important for the aggregation and emulsification stability of microemulsion droplets under different ion intensities [23]. Therefore, the effect of ionic strength (0–1000 mM NaCl) on the stability of microemulsion at pH 7 was studied.

As shown in Figure 4, at 0.1–1 mM NaCl PSO–casein–EGCG microemulsion had smaller particles and higher net negative charge than that without EGCG. The average particle size of the microemulsion sample increased with the increase in the ion concentration, which may have been caused by the high ion strength reducing the electrostatic interaction between droplets [10]. Studies on protein stabilizers have shown that the main stabilizing force is the electrostatic repulsion and spatial repulsion between proteins and oil droplets [24]. Therefore, when the electrostatic repulsion was small, these microemulsions were prone to flocculate and suspend, so the average particle size was increased. The zeta potential of the microemulsion decreased with the increase in microemulsion ion concentration, suggesting that the instability of the microemulsion at high ion strength may be due to the shielding of salt ions on the electrostatic repulsion between protein-coated oil droplets. With the increase in ion concentration, the PDI of the microemulsion was increased, indicating that the particle uniformity of the microemulsion was decreased at high salt concentration. The results in Figure 3 show the variation curve of microemulsion shear stress with shear rate under different ion concentrations (0.1–1000 mM). At a shear rate of 0–300 s⁻¹, all the microemulsions showed shear thickening behavior, and the shear stress was enhanced with the increase in shear rate. When the ion concentration was 0.1 mM, the minimum shear force was exhibited, and the shear force increased with the increase in ion concentration. Initially, microemulsion droplets were stabilized by electrostatic repulsive forces due to their negative charges [25]. However, the presence of the dissociated Na⁺ cations screened the electrical charges, reduced the electrostatic repulsion between the droplets, and resulted in aggregation [25].

At a high concentration of salt (10–1000 mM NaCl), the electrostatic repulsion was no longer strong enough and the hydrophobic interaction was dominant, so the microemulsion droplets were easily attracted to each other, resulting in droplet aggregation [26]. At a relatively low salt concentration (0.1–1 mM NaCl), the electrostatic repulsion was sufficient to overcome the interaction between van der Waals forces and hydrophobicity [24], so the microemulsion droplets did not accumulate easily. Therefore, the PSO microemulsions were stable under 0.1–1 mM NaCl.

3.3.3. Microemulsion Increased the Oxidation Stability of PSO

TBARS is an indicator of secondary oxidation product formation and correlates well with the labeling of secondary oxidation products from lipid oxidation in microemulsions [27,28]. TBARS was measured to further detect the oxidation stability of PSO microemulsions. As shown in Figure 5, after 5 days of storage, MDA increased significantly and gradually with the increase in time, which indicated that lipid oxidation occurred in both systems. In the same storage time, the MDA content of the PSO–casein complex increased from 0.062 mg/mL to 0.154 mg/mL, but the MDA content of the PSO–casein–EGCG microemulsion only increased from 0.030 mg/mL to 0.115 mg/mL. Therefore, microemulsions with EGCG have better oxidation stability than complexes without EGCG. This phenomenon may be due to EGCG in microemulsions reducing the degree of oxidation and extending the stabilization time of PSO [29]. In addition, casein as an emulsifier could prevent contact between PSO and oxygen, rendering the microemulsion system stabler [30].

3.4. PSO Microemulsion Alleviated Fat Accumulation in C. elegans

The fat of C. elegans is primarily stored in intestinal epithelial cells and the gut. ORO staining is a method used to evaluate the overall fat content of nematodes, and it has a good correlation with triglyceride (TG) content [17]. In this study, we quantified fat accumulation in nematodes using ORO staining and TG content measurement to comprehensively evaluate the lipid-lowering effects of microemulsions. As shown in Figure 6a,b, after quantifying the staining intensity, the staining results indicated that compared with the blank group, the fat accumulation in nematodes treated with the PSO–casein microemulsion and PSO–casein–EGCG microemulsion was significantly reduced by 44.32% and 52.56%, respectively (p < 0.05). Compared with nematodes treated with EGCG–casein microemulsion, those treated with the PSO–casein–EGCG microemulsion exhibited a significant reduction in lipid accumulation. TG assay results also showed a similar trend, with lipid accumulation in nematodes treated with the PSO–casein microemulsion and PSO–casein–EGCG microemulsion significantly reduced by 50.00% and 77.50%, respectively (Figure 6c). The discrepancy between ORO staining results and TG measurement results may be due to other lipophilic substances also being stained by ORO [31]. The PSO–casein–EGCG microemulsion exhibited stronger lipid-lowering effects than the PSO–casein microemulsion, indicating that EGCG can enhance the lipid-lowering effects of the PSO–casein microemulsion. In summary, these data strongly suggest that the PSO–casein–EGCG microemulsion has a significant effect in reducing fat accumulation.

Jamshidian and Rafe’s study utilized a wheat germ protein–high-methoxy pectin (DWGP–HMP) complex coacervate to encapsulate d-limonene, primarily leveraging electrostatic interactions to physically protect volatile compounds via spray-drying. While this approach is effective for volatile flavors, it lacks targeted antioxidant strategies for highly unsaturated oils like PSO, leaving inherent oxidation challenges unresolved [32]. Li et al. employed an ovalbumin–gum arabic (OVA–GA) polyelectrolyte complex to stabilize Pickering emulsions, achieving high oil content (77.7%) and processing adaptability (e.g., redispersibility, flowability) for industrial-scale production [33]. However, its functionality is limited to physical barriers without conferring additional bioactivity [33]. In contrast, our PSO–casein–EGCG microemulsion not only enhances oxidative stability through EGCG but also demonstrates significant lipid-lowering effects in C. elegans. This dual advantage—combining oxidation control for unsaturated oils with bioactive functionality—aligns more closely with the demands of functional food development.

3.5. PSO Microemulsion Reduced the Size of Lipid Droplets in ZXW618 Strain

Lipid droplets are eukaryotic organelles that store neutral fats such as triacylglycerol and cholesterol esters [17]. Lipid droplets play an important role in energy utilization and storage in mammals and C. elegans [18]. Many of the fat-regulating genes found in C. elegans are homologous with mammals. Strain ZXW618 is a transgenic worm that fuses green fluorescent protein (GFP) fusion to express lipid droplet marker protein DHS-3, and lipid droplets can be clearly observed in strain ZXW618 [17]. Since the PSO–casein–EGCG microemulsion reduced fat accumulation in nematodes, its effect on the lipid droplet size of nematodes was studied. Compared with the nematodes in the control group, no significant difference was detected in the average number of lipid droplets (p > 0.05, Figure 7), but a 33.53% decrease and 41.23% decrease in lipid droplet size was observed in those treated with PSO–casein and PSO–casein–EGCG microemulsions, respectively. These results indicated that PSO microemulsions can reduce fat accumulation in the form of lowering lipid droplet size in nematodes. Therefore, microemulsions could enhance the lipid-lowering properties of PSO and are effective in protecting the stability of PSO. In addition, studies have shown that curcumin-loaded microemulsion enhanced bioaccumulation and reduced fat content of C. elegans [34]. Vitamin E contained in almond oil or neem oil has good stability and enhanced anti-inflammatory activity [35], indicating that developing a transport system for a bioactive compound could improve stability and utilization and better utilize their activity.

The ability of PSO–casein–EGCG microemulsions to reduce lipid droplet size in ZXW618 mutant nematodes suggests potential applicability to mammalian systems, where smaller lipid droplets correlate with improved lipid metabolism [36,37]. This positions these microemulsions as promising candidates for lipid-lowering functional foods. The EGCG–casein complex not only stabilizes microemulsions but also enhances bioactivity through synergistic antioxidant and lipid-lowering effects. This model could be adapted for co-delivering other lipophilic bioactives (e.g., vitamin D/E or polyphenols), enabling personalized nutrition strategies.

While the findings on PSO–casein–EGCG microemulsions are encouraging, several limitations of this study should be noted. Firstly, although the fatty acid composition of PSO was characterized by GC–MS (Table 3), a more detailed analysis of minor bioactive constituents—such as tocopherols, phytosterols, and polyphenols—was not conducted. Inclusion of such data would offer deeper insights into the antioxidant profile and nutritional quality of the PSO used. Future investigations should encompass comprehensive phytochemical characterization to better elucidate the composition–function relationships of the oil. Secondly, in the C. elegans assays, a free PSO (unencapsulated) control was not evaluated. While the microemulsion system showed significant reduction in lipid accumulation and droplet size, the absence of a direct comparison with unencapsulated PSO restricts definitive conclusions regarding the contribution of the delivery system per se to the observed efficacy. Further studies should include free PSO treatments to decouple the effects of the encapsulation from those of the native oil. Lastly, several practical challenges must be overcome to facilitate real-world application. Scaling production and managing costs represent considerable obstacles. High-pressure homogenization, essential for achieving nanoscale droplets, is energy-intensive and economically challenging at industrial scales. Future efforts should focus on optimizing process parameters or developing alternative low-energy emulsification technologies. Stability in complex food environments also requires further assessment: although the microemulsions remained stable within pH 5–9 and under low-ionic-strength conditions, their behavior in highly acidic systems (e.g., citrus beverages, pH < 4) or high-salt products (e.g., sauces with NaCl > 1 mM) remains unknown. Such conditions could compromise the integrity of the EGCG–casein complex and thus necessitate matrix-specific stability evaluations. Addressing these limitations in subsequent research will be crucial to affirming the practical potential of PSO–casein–EGCG microemulsions.

4. Conclusions

The preparation of PSO-casein-EGCG microemulsions was performed in this study. The PSO microemulsion had a particle size of 361.23 ± 14.85 nm, a zeta potential of −20.77 ± 0.68 mV, and a PDI of 0.17 ± 0.07, indicating a uniform microemulsion system. Subsequently, the physical and chemical stability of the microemulsion was investigated. The prepared microemulsion was stored at room temperature for 5 days, with NaCl concentrations ranging from 0.1 mM to 1 M and pH values between 5 and 8, showing good stability. Additionally, the presence of EGCG enhanced the oxidative stability of PSO–casein–EGCG microemulsions compared to PSO–casein microemulsions under the same storage conditions. Finally, treatment with PSO–casein–EGCG microemulsions significantly reduced lipid droplet size in nematodes, indicating that these microemulsions have good fat-reducing effects.

In summary, these results indicate that PSO–casein–EGCG microemulsions are a feasible strategy for overcoming the limitations of PSO application. They exhibit good physical and chemical stability over a 5-day storage period. Experimental results show that microemulsions significantly reduce lipid accumulation and shrink lipid droplet size in a nematode model.