Applicability of transglycosylated stevia for oil-in-water submicron emulsions by high-pressure homogenization

Abstract

An oil-in-water emulsion with submicron particle size was prepared using a high-pressure homogenizer (HPH) in the presence of transglycosylated stevia (Stevia-G). Before HPH processing, the emulsions were coarse and microscale in particle size. Although Stevia-G has weak surface-active properties, HPH enabled the preparation of submicron emulsions. HPH processing conditions affected the particle size and encapsulation efficiency of flavanone. The particle size of emulsions did not change upon storage and the encapsulation efficiency of flavanone was 85.1 ± 2.9% at an operating pressure of 100 MPa. The HPH-processed emulsions were successfully powdered by freeze-drying with trehalose and exhibited similar particle sizes and encapsulation efficiencies to those prior to freeze-drying. After digestion of the emulsions in simulated gastrointestinal fluid, the HPH-processed emulsions showed higher flavanone concentrations compared to flavanone powder. HPH reduces the oil droplet size and Stevia-G acts as an emulsifier to stabilize submicron emulsions.

Introduction

Emulsions are widely used in the food industry to improve the solubility of poorly water-soluble compounds and for chemical stabilization (Jacobsen et al., 2008; Yoo et al., 2010). Polyphenols and carotenoids, which have poor water solubility and chemical stability, have been used in emulsions to improve these drawbacks (Chen et al., 2017; Lu et al., 2016). Emulsions are defined as immiscible liquid dispersions comprised of two or more phases. Oil-in-water emulsions, in which the oil phase is dispersed in an aqueous solution, are prepared using high-pressure homogenizers (HPH) and membrane emulsification (Fernandez-Avila and Trujillo, 2016; Hebishy et al., 2017; Nakashima et al., 2000). In the HPH, the samples are passed through a narrow gap under high pressure in the range of 100–200 MPa. The HPH supplies energy to increase the water/oil interfacial area via cavitation, shear stress, and high turbulence. The HPH can reduce the droplet size to <1 µm and improve the storage stability of emulsions by reducing the creaming rate. The operating pressure and pass time in the HPH are critical parameters used to control the particle size and storage stability of the resulting emulsions. Therefore, optimization studies on the HPH operating parameters are important. Selection of the emulsifier is also an important experimental parameter in the emulsification process. Surfactants comprising both hydrophobic and hydrophilic components are generally used as emulsifiers (Ma et al., 2018). Sugar-based surfactants, in which the sugar moiety is the hydrophilic component, are one of the common surfactants used for emulsification (Ismail et al., 2001; Neta et al., 2015). Sugar-based surfactants are divided into two types, those extracted from plants and those prepared using chemical synthesis. Saponins, which are natural surfactants extracted from plants, are predominantly glycosides with sugar chains attached to the aglycone. Yang et al. reported that Quillaja saponin exhibited excellent emulsification properties under various pH conditions, salt concentrations, and temperatures (Yang et al., 2013). Sugar esters are mainly synthesized by adding sugar moieties to the carbon chain. Sucrose monopalmitate can be used as a food additive to prepare nano- and microemulsions, which have been widely applied in the food, cosmetic, and pharmaceutical industries (Rao and McClements, 2011).

Stevia is a herb belonging to the Compositae family, which is estimated to comprise 150–300 species (Grashoff et al., 1972; King and Robinson, 1967). Stevia rebaudiana, which is commonly known as sweet leaf, sugar leaf, or simply stevia, is widely grown for its sweet leaves. As a sweetener and sugar substitute, the taste of stevia has a slower onset and longer duration than that of sugar. Steviosides have been used as natural sweeteners for 20 years and no significant adverse effects have been reported to date. α-Glucosyl stevia (Stevia-G) is synthesized from stevia upon the addition of a sugar moiety using glycosyltransferase, and has a sweeter taste and higher solubility than stevia. We have previously reported that Stevia-G shows surface-active properties and forms a micelle structure at concentrations >10 mg/mL, indicating that Stevia-G may also act as a sugar-based surfactant (Uchiyama et al., 2010; Zhang et al., 2014). Stevia-G enhances the solubility of poorly water-soluble compounds by solubilizing them within micelles and improves the oral absorption of these compounds (Tozuka et al., 2013). In addition to its use as a solubilizer or taste improver, Stevia-G can be applied as an emulsifier due to its surface-active properties; however, few studies have used Stevia-G as an emulsifier. The reason for this is the weak surface-active properties of Stevia-G compared to those of conventional emulsifiers, such as sugar esters.

The purpose of this study was to examine the applicability of Stevia-G as an emulsifier. Nanosized emulsions were prepared using Stevia-G as an emulsifier and an HPH. The effects of operating pressure and pass time on the physicochemical properties of the resulting emulsions were investigated. Stevia-G and olive oil were used as the emulsifier and oil, respectively. Flavanone was used as a functional model compound with poor water solubility. The storage stability of the HPH-processed emulsions was evaluated upon storage at 4 °C for 2 weeks. Powders containing the HPH-processed emulsions were prepared via a freeze-drying method using a cryoprotectant. Furthermore, the re-dispersibility and solubility of the freeze-dried powders were evaluated using distilled water and simulated gastrointestinal fluids, respectively.

Materials and Methods

Materials    Stevia-G was kindly gifted by Toyo Sugar Refining Co., Ltd. (Tokyo, Japan). Olive oil was provided by KANEDA Co., Ltd. (Tokyo, Japan). Flavanone and trehalose were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ammonium carbonate, calcium chloride dihydrate, magnesium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, sodium chloride, gall powder, and pepsin from porcine stomach were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Pancreatin was purchased from Sigma-Aldrich (Tokyo, Japan). All other chemicals and solvents were of reagent grade or high-performance liquid chromatography (HPLC) grade and used without further purification.

Preparation of oil-in-water emulsions without flavanone    Oil-in-water emulsions without flavanone were prepared by alterations in Stevia-G contents. The concentration of Stevia-G was less than the critical micelle concentration, ensuring that Stevia-G itself does not form micelles. Stevia-G (0.5–3 g) was dissolved in distilled water (300 mL). Olive oil (1.0 g) was added to the solution containing Stevia-G. Coarse emulsions were prepared using a homogenizer (T 25 digital ULTRA-TURRAX®, IKA®, Freiburg, Germany) operated at 8 000 rpm for 5 min at 25 ± 5 °C. The coarse emulsions were further homogenized using an HPH (NANOMEISTER, OKAWARA MFG., CO., LTD., Shizuoka, Japan) under conditions of one pass time and at 100 MPa. The emulsions were cooled to below 50 °C using a water circulation apparatus (CA-1115, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) in the HPH.

Preparation of oil-in-water emulsions containing flavanone    Oil-in-water emulsions containing flavanone were prepared with a fixed additive content. Stevia-G (1.5 g) was dissolved in distilled water (300 mL). Olive oil (1.0 g) solubilized flavanone (0.1 g) was added to the solution containing Stevia-G. Coarse emulsions were prepared using a homogenizer operated at 8 000 rpm for 5 min at 25 ± 5 °C. The coarse emulsions were further homogenized using an HPH under the conditions listed in Table 1. The emulsions were cooled to below 50 °C using a water circulation apparatus in the HPH.

Table 1. Operating conditions for the high-pressure homogenizer to prepare an emulsion including flavanone.

Formulation	F1	F2	F3	F4	F5	F6	F7
Operating pressure (MPa)	50	50	100	100	100	150	150
Pass time	1	5	1	3	5	1	5

Particle size measurements    Particle sizes <1 µm were measured on a Nanotrac UPA instrument using dynamic light scattering (UPA-UT151, MicrotracBEL, Osaka, Japan), while particle sizes > 1 µm were measured using laser diffraction analysis (HRA-9320, MicrotracBEL). All measurements were performed at 25 ± 5 °C with a waiting time of 30 s and a measurement time of 60 s. The D₁₀, D₅₀ and D₉₀ values of each measurement from the prepared three samples were reported. The parameter D₁₀ is the point on the size distribution curve below which 10% of the particles fall. The parameter D₅₀ is the point on the size distribution curve that divides equal amounts of smaller and larger particles. The parameter D₉₀ is the point on the size distribution curve below which 90% of the particles fall. The span value of average median diameter was calculated using Eq. 1:   

Encapsulation efficiency of flavanone    Each emulsion was centrifuged at 2 000g for 10 min (Model 2410, KUBOTA Corporation Co., Ltd., Tokyo, Japan). The concentrations of flavanone in the supernatant and the flavanone encapsulated in the emulsion were determined using HPLC (SPD-10A, Shimadzu Co., Ltd., Kyoto, Japan) on a COSMOSIL 5C18-MS-II packed column (5 µm, 150 mm × 4.6 mm, Nacalai Tesque, Kyoto, Japan). The mobile phase consisted of acetonitrile and 0.1% phosphoric acid (60/40, v/v). The flow rate was controlled at 1.0 mL/min using an injection volume of 10 µL. Flavanone was eluted at 40 °C and the absorbance was quantified at 254 nm. The encapsulation efficiency of flavanone was determined using Eq. 2:   

Storage stability of emulsions    Each emulsion was stored in a water bath at 4 °C for 2 weeks. The particle size of the emulsion and the encapsulation efficiency of flavanone were evaluated after 2 weeks, as described in the methods section for particle size measurements and encapsulation efficiency of flavanone.

Preparation of freeze-dried powders    The resulting emulsion was freeze-dried using trehalose. A 3x volume of trehalose with respect to the emulsion components, except water, was added and the mixture was frozen at −40 °C for 30 min in a circulation bath (PFR-1000, Tokyo Rikakikai Co., Ltd.). Lyophilization of the emulsions with/without trehalose was performed at ∼5 Pa for 24 h using a freeze-dryer (FDU-830, Tokyo Rikakikai Co., Ltd.). The freeze-dried powders were redispersed in distilled water. The particle size of the emulsions and the encapsulation efficiency of flavanone were evaluated after 2 weeks, as described in the methods section for particle size measurements and encapsulation efficiency of flavanone.

Solubility studies    Solubility studies were carried out in 40 mL of simulated gastric fluid and simulated intestinal fluid at 37 °C and 100 strokes per minute using a cool bath shaker (ML-10F, Taitec Corporation, Saitama, Japan). The simulated gastric fluid (1 L) was composed of potassium chloride (0.821 g), potassium dihydrogen phosphate (0.313 g), sodium hydrogen carbonate (1.621 g), sodium chloride (1.381 g), magnesium chloride (0.028 g), ammonium carbonate (0.027 g), calcium chloride dihydrate (0.134 g), and pepsin (16 g), which was adjusted to pH 3.0 using hydrochloric acid. The simulated intestinal fluid (1 L) was composed of potassium chloride (0.507 g), potassium dihydrogen phosphate (0.109 g), sodium hydrogen carbonate (7.14 g), sodium chloride (2.246 g), magnesium chloride (0.067 g), ammonium carbonate (0.027 g), calcium chloride dihydrate (0.177 g), gall powder (8.881 g), and pancreatin (1.101 g), which was adjusted to pH 7.0 using hydrochloric acid and sodium hydroxide. The freeze-dried powders prepared with trehalose were added to 20 mL of simulated gastric fluid containing flavanone (12 mg). After 1 h, the simulated intestinal fluid was added to the simulated gastric fluid, and the mixture was adjusted to pH 7.0 using 1 M sodium hydroxide. A 1-mL portion of the sample was removed at 4 h after changing the pH to 7.0 and centrifuged at 2 000g for 10 min (Model 2410, KUBOTA Corporation Co., Ltd.). The concentration of flavanone was determined using HPLC, as described in the methods section for encapsulation efficiency of flavanone.

Statistical analysis    The results of each study are expressed as mean ± standard deviation (SD) of three experiments.

Results and Discussion

The effect of Stevia-G amount on the emulsification of olive oil was firstly investigated with HPH processing. Before HPH processing, the emulsion was processed using a homogenizer at 8 000 rpm for 5 min. The D₅₀ value of all emulsions was greater than 10 µm. The coarse emulsion was processed using an HPH under the conditions of one pass time and 100 MPa. The particle sizes of the emulsions prepared with HPH are listed in Table 2. Increases in Stevia-G amount resulted in decreased particle size of the emulsion after preparation. The emulsion containing Stevia-G at 1.0 g or higher showed almost the same particle size. The Stevia-G amount was related to the particle size of the emulsion after storage at 4 °C for 2 weeks. The particle size of the emulsion prepared with Stevia-G at 0.5 or 0.75 g greatly increased after storage. Furthermore, the particle size of the emulsion prepared with 1.0 g Stevia-G increased somewhat after storage. The addition of 1.5 g or higher Stevia-G was effective for the stable emulsification of olive oil. Stevia-G, which has both hydrophobic and hydrophilic parts in its chemical structure, shows surface-active properties in aqueous media. Therefore, Stevia-G could be oriented at the interface between oil and water. The orientation of Stevia-G at the interface enhanced the dispersion to the water of oil droplets sheared by HPH processing. On the other hand, a large amount of Stevia-G was necessary to achieve stable emulsification of olive oil, as Stevia-G has weak surface-active properties compared to conventional emulsifiers (Yukuyama et al., 2019). From these results, a Stevia-G amount of 1.5 g was selected in the subsequent study containing flavanone.

Table 2. Change in the particle size of the emulsion without flavanone after preparation and storage at 4 °C for 2 weeks. Each point represents the mean ± SD (n =3).

			Particle size (µm)
Stevia-G (g)	Olive oil (g)	Water (g)	After preparation				After 2 weeks
			D10	D50	D90	Span	D10	D50	D90	Span
0.5	1.0	300	0.125 ± 0.011	0.226 ± 0.009	0.447 ± 0.064	1.42	0.138 ± 0.007	0.275 ± 0.018	0.747 ± 0.058	2.21
0.75	1.0	300	0.112 ± 0.018	0.191 ± 0.013	0.359 ± 0.027	1.30	0.113 ± 0.013	0.211 ± 0.036	0.558 ± 0.043	2.11
1.0	1.0	300	0.080 ± 0.007	0.152 ± 0.004	0.308 ± 0.019	1.50	0.074 ± 0.007	0.180 ± 0.011	0.363 ± 0.035	1.60
1.5	1.0	300	0.070 ± 0.010	0.148 ± 0.008	0.285 ± 0.032	1.45	0.071 ± 0.021	0.147 ± 0.017	0.281 ± 0.017	1.43
3.0	1.0	300	0.069 ± 0.006	0.151 ± 0.007	0.268 ± 0.021	1.36	0.073 ± 0.003	0.146 ± 0.010	0.274 ± 0.018	1.38

The emulsion containing flavanone was prepared by changing the condition of HPH processing. Before HPH processing, the emulsion was processed using a homogenizer at 8 000 rpm for 5 min, and the particle size distribution of the prepared emulsions is shown in Figure 1. The prepared samples showed a wide particle size distribution and formed a coarse emulsion with a median particle size (D₅₀) of 11.53 µm. The coarse emulsion prepared by a homogenizer was HPH processed under the conditions shown in Table 1. The particle sizes of the HPH-processed emulsions are listed in Table 3. All HPH-processed emulsions showed a D₅₀ value of < 0.2 µm. Furthermore, an increase in the operating pressure and pass time resulted in a reduction in the particle size. When the processed emulsions were stored at 4°C for 2 weeks, no significant changes were observed in particle size. There are several reports on the relationship between HPH operating conditions and particle size. Mao et al. reported on the mathematical relationship between the operating pressure and particle size of an emulsion (Mao et al., 2010). The particle size of the emulsion gradually decreased upon increasing the operating pressure. Ruiz et al. investigated the effect of the pass time on the particle size of an emulsion (Ruiz-Montañez et al., 2017). An increase in the pass time also decreased the particle size of the emulsion. In the HPH process, an increase in the operating pressure and pass time results in a larger supply of mechanical energy to the oil droplets, which creates a new water/oil interface. This mechanical energy is used to decrease the size of the oil droplets. Although the sheared oil droplets are very unstable up to a nanoscale range due to their large surface area in water, the surfactant can be adsorbed onto the surface of the sheared oil droplets, stabilizing the resulting emulsion with a small particle size. In this study, a submicron emulsion was prepared using Stevia-G as an emulsifier and the particle size of the prepared emulsion was maintained upon storage. This result suggests that Stevia-G can be used as an emulsifier to prepare submicron emulsions suitable for HPH processing.

Fig. 1.

Particle size distribution of the emulsion before processing using a high-pressure homogenizer.

Table 3. Change in the particle size of the emulsion including flavanone after preparation and storage at 4 °C for 2 weeks. Each point represents the mean ± SD (n =3).

Formulation	Particle size (µm)
	After preparation				After 2 weeks
	D10	D50	D90	Span	D10	D50	D90	Span
F1	0.100 ± 0.017	0.207 ± 0.015	0.735 ± 0.103	3.07	0.082 ± 0.021	0.198 ± 0.040	0.479 ± 0.077	2.01
F2	0.073 ± 0.014	0.165 ± 0.021	0.348 ± 0.039	1.67	0.074 ± 0.027	0.164 ± 0.028	0.423 ± 0.046	2.13
F3	0.067 ± 0.010	0.141 ± 0.025	0.307 ± 0.028	1.70	0.064 ± 0.020	0.127 ± 0.026	0.310 ± 0.036	1.94
F4	0.071 ± 0.011	0.151 ± 0.029	0.291 ± 0.025	1.46	0.071 ± 0.027	0.156 ± 0.017	0.301 ± 0.033	1.47
F5	0.070 ± 0.022	0.145 ± 0.019	0.291 ± 0.036	1.52	0.050 ± 0.007	0.115 ± 0.011	0.248 ± 0.027	1.72
F6	0.071 ± 0.010	0.150 ± 0.017	0.293 ± 0.024	1.48	0.073 ± 0.011	0.133 ± 0.028	0.305 ± 0.028	1.74
F7	0.067 ± 0.010	0.135 ± 0.015	0.235 ± 0.021	1.24	0.058 ± 0.008	0.118 ± 0.019	0.231 ± 0.022	1.47

Figure 2 shows the encapsulation efficiency of flavanone in the HPH-processed emulsions after their preparation and storage at 4 °C for 2 weeks. The processing conditions affect the entrapment of flavanone in the oil phase of the emulsion. The leakage and precipitation of flavanone from the emulsions were observed after storage at 4 °C for 2 weeks. When the coarse emulsions processed at 50 and 150 MPa were stored at 4 °C for 2 weeks, the encapsulation efficiency of flavanone decreased compared to that prepared at 100 MPa after storage at 4 °C for 2 weeks. It is important to maintain flavanone in the oil phase after its preparation and preservation, as flavanone shows low oral absorption due to its poor solubility. Li et al. reported that the stirring speed and time can induce a decrease in the encapsulation efficiency during the homogenization process (Li et al., 2017). The energy provided by the homogenization process generates heat in the emulsion system, leading to an increase in temperature. The elevated temperature then decreases the viscosity of the dispersion to obtain smaller particle size droplets. The decreased oil viscosity may induce the release of the encapsulated components from the oil phase. Stevia-G has weak surface-active properties compared to commonly used surfactants. Although Stevia-G could be oriented at the interface between oil and water through HPH processing, the degree of orientation may depend on the processing conditions. At the highest operating pressure of 150 MPa, the decrease in oil viscosity may induce the desorption of Stevia-G from the oil phase. The orientation of the decreased Stevia-G resulted in the leakage of flavanone from the oil phase. At the lowest operating pressure of 50 MPa, the new interface formation between oil and water may be insufficient due to its low shear force, as shown by the larger particle size after storage or preparation. In the case of Stevia-G, oleic acid, and flavanone systems, an operating pressure of 100 MPa was estimated to be the optimal operating condition in terms of particle size and encapsulation efficiency.

Fig. 2.

Change in the encapsulation efficiency of flavanone after preparation and storage at 4 °C for 2 weeks. Each point represents the mean ± SD (n =3).

Although the particle size of the HPH-processed emulsions did not change after storage, the leakage and precipitation of flavanone were observed. When considering the oral absorption of flavanone, flavanone should be encapsulated in emulsions because it is a poorly water-soluble compound. Therefore, freeze-dried particles were prepared with and without trehalose as a cryoprotectant. Table 4 and Figure 3 show the particle size of the redispersed emulsions and the encapsulation efficiency of flavanone after freeze-drying. The particle size of the emulsion without trehalose increased after freeze-drying. In addition, leakage of flavanone from the oil phase was observed in the freeze-dried particles prepared without trehalose. These results suggest that the oil-in-water emulsion collapsed and the oil droplets merged during the freeze-drying process. On the other hand, the addition of trehalose prevented the collapse of the emulsion during the freeze-drying process. The redispersed emulsions showed almost the same particle size and encapsulation efficiency as the emulsions prior to freeze-drying. Sugar alcohols such as trehalose are widely used as cryoprotectants to prevent the collapse of emulsions during the freeze-drying process (Wu et al., 2019). Freeze-dried particles of the HPH-processed emulsions were successfully prepared upon the addition of trehalose.

Table 4. Particle size of the redispersed emulsion after freeze drying without and with trehalose. Each point represents the mean ± SD (n =3).

Formulation	Particle size (µm)
	Without cryoprotectant				With trehalose
	D10	D50	D90	Span	D10	D50	D90	Span
F1	1.288 ± 0.489	2.757 ± 0.698	5.941 ± 1.968	1.69	0.102 ± 0.011	0.199 ± 0.030	0.575 ± 0.052	2.38
F2	1.430 ± 0.308	3.543 ± 0.456	16.57 ± 8.016	4.27	0.095 ± 0.023	0.190 ± 0.027	0.440 ± 0.073	1.82
F3	1.236 ± 0.251	2.666 ± 0.596	5.841 ± 2.146	1.73	0.072 ± 0.019	0.158 ± 0.018	0.383 ± 0.036	1.97
F4	1.136 ± 0.328	2.835 ± 0.528	5.498 ± 2.337	1.54	0.082 ± 0.017	0.154 ± 0.018	0.318 ± 0.048	1.53
F5	1.105 ± 0.241	2.471 ± 0.448	6.952 ± 3.310	2.37	0.077 ± 0.013	0.170 ± 0.021	0.312 ± 0.026	1.38
F6	1.033 ± 0.405	2.327 ± 0.434	4.991 ± 1.310	1.70	0.069 ± 0.011	0.149 ± 0.013	0.322 ± 0.037	1.70
F7	0.998 ± 0.397	2.042 ± 0.699	4.316 ± 1.884	1.62	0.064 ± 0.009	0.122 ± 0.011	0.227 ± 0.031	1.34

Fig. 3.

The encapsulation efficiency of flavanone in the redispersed emulsion after freeze drying without and with trehalose. Each point represents the mean ± SD (n =3).

Therefore, for the flavanone encapsulated in the emulsion to be absorbed by the small intestine after oral consumption, the emulsion must first be digested in the gastrointestinal fluid and its effective components released from the digested emulsion. The released components are solubilized in the micellar structures formed by the emulsifier and taurocholic acid. The solubilized components are then absorbed by the small intestine. Therefore, it is important to study the concentration of the dissolved components in gastrointestinal fluid containing taurocholic acid after digestion of the emulsion. Figure 4 shows the dissolved flavanone from the redispersed emulsion in simulated gastrointestinal fluid containing taurocholic acid and enzyme. Flavanone powder shows a low dissolution ratio because of its poor solubility in gastrointestinal fluid. When the freeze-dried particles were placed in simulated intestinal fluid, the dissolved flavanone was increased compared to the flavanone powder. The emulsions processed at > 100 MPa tended to show a higher dissolution ratio. Table 4 shows that the emulsions processed at > 100 MPa had smaller particle sizes compared to those prepared at 50 MPa. Emulsions with smaller particle sizes have a large surface area and high digestibility in simulated gastrointestinal fluid. The enhanced dissolution ratio observed in the simulated gastrointestinal fluid is thought to facilitate digestion and solubilization into the micellar phase.

Fig. 4.

Dissolved flavanone concentration from the freeze-dried particles in simulated gastrointestinal fluid. Each point represents the mean ± SD (n =3).

Conclusions

An oil-in-water submicron emulsion was successfully prepared using an HPH and Stevia-G. Although Stevia-G has weak surface-active properties compared to conventional sugar-based surfactants, it can be applied as an emulsifier to stabilize emulsions with submicron particle sizes. When the HPH operating conditions were investigated in terms of the operating pressure and pass time, the operating conditions were found to affect the particle size of the emulsion and the encapsulation efficiency of flavanone. The particle size showed a tendency to decrease with increases in the operating pressure and pass time. On the other hand, processing at the highest operating pressure of 150 MPa resulted in decreased encapsulation efficiency after storage at 4 °C for 2 weeks. Freeze-dried particles with high redispersibility were prepared using trehalose. Oil-in-water emulsions with the same particle size and encapsulation efficiency observed prior to freeze drying were reformed after dispersing the freeze-dried particles. The freeze-dried particles showed improved solubility in simulated gastrointestinal fluid, indicating that the nanosized oil-in-water emulsion with Stevia-G can release flavanone via emulsion digestion. This study describes a new method to prepare submicron emulsions using an HPH and Stevia-G.

Acknowledgements    The authors are grateful to OKAWARA MFG., CO., LTD. (Shizuoka, Japan) for the loan of the NANOMEISTER instrument. We are also grateful to Toyo Sugar Refining Co., Ltd. (Tokyo, Japan) for gifting the transglycosylated stevia used in this study.

Conflict of interest    There are no conflicts of interest to declare.

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