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Journal of Oleo ScienceCopyright ©2015 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess14229J. Oleo Sci. 64, (4) 405-413 (2015)

O/W Nano-Emulsion Formation Using an Isothermal Low-Energy Emulsification Method in a Mixture of Polyglycerol Polyricinoleate and Hexaglycerol Monolaurate with Glycerol SystemSatoshi Wakisaka1, 2, Takahisa Nishimura2 and Shoichi Gohtani3＊1 Department of Food Science, The United Graduate School of Agricultural Sciences, Ehime University (2393 Ikenobe, Miki-cho, Kagawa

761-0795, JAPAN)2 Research and Development Division, Glico Nutrition Co., Ltd. (4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, JAPAN)3 Department of Applied Biological Science, Faculty of Agriculture, Kagawa University (2393 Ikenobe, Miki-cho, Kagawa 761-0795, JAPAN)

1 INTRODUCTIONNano-emulsions are emulsions whose droplet diameter

typically falls in the range of 20–200 nm1）. The size of the droplets in nano-emulsions is often much smaller than the wavelength of light（d << λ）; therefore, nano-emulsions do not scatter light strongly, making them transparent or translucent2）. Nano-emulsions also exhibit significantly im-proved emulsion stability against creaming and flocculation due to the substantially reduced rates of gravitational sepa-ration and enhanced Brownian motion of oil droplets3）. Finally, nano-emulsion technology has been applied in fab-ricating encapsulating systems for functional compounds because it prevents degradation and improves bioavailabili-ty4）.

＊Correspondence to: Shoichi Gohtani, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kagawa 761-0795, JAPANE-mail: gohtani@ag.kagawa-u.ac.jpAccepted December 19, 2014 (received for review October 10, 2014)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

In general, nano-emulsions can be achieved using either high-energy emulsification methods or low-energy emulsi-fication methods. High-energy emulsification methods involve an intensive energy input using a high-shear stirrer, a high-pressure homogenizer or ultrasound generators1）. Alternatively, low-energy emulsification methods that utilize thermodynamic driving forces have been developed, enabling the formation of nano-emulsions with minimal energy input of mechanical energy5－10）. There is interest in using lower energy techniques in the emulsion formation process because of economic benefits. Low-energy emulsi-fication methods can be broadly categorized as either thermal or isothermal methods11）. Thermal methods rely on emulsion formation resulting from changes in surfactant

Abstract: We investigated how phase behavior changes by replacing water with glycerol in water/mixture of polyglycerol polyricinoleate (PGPR) and hexaglycerol monolaurate (HGML) /vegetable oil system, and studied the effect of glycerol on o/w nano-emulsion formation using an isothermal low-energy method. In the phase behavior study, the liquid crystalline phase (Lc) + the sponge phase (L3) expanded toward lower surfactant concentration when water was replaced with glycerol in a system containing surfactant HLP (a mixture of PGPR and HGML). O/W nano-emulsions were formed by emulsification of samples in a region of Lc + L3. In the glycerol/surfactant HLP/vegetable oil system, replacing water with glycerol was responsible for the expansion of a region containing Lc + L3 toward lower surfactant concentration, and as a result, in the glycerol/surfactant HLP/vegetable oil system, the region where o/w nano-emulsions or o/w emulsions could be prepared using an isothermal low-energy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller than that in the water/surfactant HLP/vegetable oil system. Therefore, glycerol was confirmed to facilitate the preparation of nano-emulsions from a system of surfactant HLP. Moreover, in this study, we could prepare o/w nano-emulsions with a simple one-step addition of water at room temperature without using a stirrer. Thus, the present technique is highly valuable for applications in several industries.

Key words: nano-emulsion, isothermal low-energy emulsification, sponge phase, liquid crystalline, glycerol

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properties with temperature, whereas isothermal methods rely on emulsion formation resulting from changes in local system composition at a fixed temperature12）. Spontaneous emulsification and phase inversion composition methods fall into the category of isothermal methods13）, while the phase inversion temperature method is an example of a thermal method14, 15）.

In our previous study, we have demonstrated that nano-emulsions suitable for use in food systems can be prepared using an isothermal low-energy emulsification method, consisting of stepwise addition of water to the phase of the water/mixture of polyglycerol polyricinoleate（PGPR）and polyglycerol fatty acid ester（PGFA）/vegetable oil at a con-stant temperature（25℃）with moderate mixing using a stirrer operated at a rotational speed around 300 rpm16）. In comparing the phase diagram of the ternary system and the droplet size in the resulting emulsions, it was found that o/w nano-emulsions with droplet sizes as small as 50 nm were formed by emulsifying from either a single sponge phase（L3）or a two-phase region, liquid crystalline phase（Lc）＋L3. These results indicate that L3 or Lc, or both, is necessary to form an o/w nano-emulsion for PGPR and PGFA mixtures used as surfactant. Therefore, it can be ex-pected that finding specific conditions to expand L3 or Lc in the phase diagram for PGPR and PGFA mixtures may be applied to prepare an o/w nano-emulsion using an isother-mal lower-energy emulsification method.

It is known that the presence of cosolvents（such as alco-hols and polyols）affect the phase behavior of surfactants dispersed in aqueous solutions and hence the outcome of low-energy emulsification is also affected6, 17－19）. Among the cosolvents, glycerol is mostly used in industries such as pharmaceutical, cosmetic, food and chemical. Adding glyc-erol to aqueous solutions has been shown to alter the inter-facial tension, optimum curvature and solubility character-istics of both ionic and non-ionic sufactants20－24）. A number of studies have investigated the influence of glycerol and similar cosolvents on various types of emulsion-based systems19, 20, 24）. Suzuki et al. showed that the presence of an appropriate amount of glycerol enhanced the rigidity of bilayers forming a lamellar liquid crystal in an L-arginine hexyldecyl phosphate/water/glycerol system, thus promot-ing the formation of an oil-in-liquid crystal emulsion5）. As a result, they developed a liquid crystal emulsification tech-nique known as the low-energy emulsification method. However, the effects of glycerol on a mixture of PGPR and PGFA systems have not yet been investigated.

The objective of this research is to investigate how phase behavior changes by replacing water with glycerol in water/mixture of PGPR and hexaglycerol monolaurate（HGML）/vegetable oil systems, and to determine the effect of incor-porating glycerol in mixtures of PGPR and HGML on nano-emulsion formation using an isothermal low-energy method. In addition, unlike in our previous study where we

added water step by step to the prepared phase while stir-ring16）, in this study, we attempted to prepare nano-emul-sions with a simple one-step addition of water to the pre-pared phase without using a stirrer.

2 EXPERIMENTAL PROCEDURES2.1 Materials

PGPR（commercial name SY-Glyster CRS-75）, HGML（commercial name SY-Glyster ML-500）were purchased from Sakamoto Yakuhin Kogyo Co., Ltd. Glycerol was pur-chased from Wako Pure Chemical Industries, Ltd. Vegeta-ble oil（consisting of soybean oil and rapeseed oil）produced by Nisshin Oillio group Ltd. was purchased from conven-tional supermarket. All materials were used without further purification. Purified water was prepared using an E-pure system（Dubuque, USA）.

We used a mixture of PGPR with HGML（weight ratio 1:1）in this study. The mixtures of PGPR and HGML are ab-breviated as surfactant HLP.

2.2 Methods2.2.1 Determination of the phase diagram

Test tubes containing mixtures with the desired compo-sitions were repeatedly shaken using a vortex mixer and then stored at 25℃. The formation of liquid crystals was confirmed using polarizing plates placed on either side of the test tube. If the liquid crystalline phase was formed, light passed through the plates due to the birefringence of the liquid crystalline. Detailed phases were identified using polarized light microscopy（OLYMPUS, BH-2）and small-an-gle X-ray scattering（SAXS）techniques.2.2.2 Small-angle X-ray scattering

SAXS measurements were performed on a Nano-Viewer SAXS instrument（Rigaku Co., Tokyo, Japan）equipped with a PILATUS at an applied voltage and filament current of 40 kV and 30 mA, respectively. The wavelength of the radia-tion source was 0.154 nm.2.2.3 Preparation of an O/W emulsion

Vegetable oil, water or glycerol and the emulsifier mix-tures previously described in section 2.1 were mixed at the desired weight ratios to prepare mixtures before emulsifi-cation. The mixtures before emulsification were put in test tubes, and water was added to the test tubes in one step such that the final aqueous phase concentration was 99 wt％. The test tubes were then gently shaken by hand at 25℃ to prepare an o/w emulsion.2.2.4 Measurement of the particle size distribution and

polydispersityParticle size distribution and polydispersity index（PDI）

were determined using a dynamic light scattering device（FPER-1000; Otsuka Electronics, Osaka, Japan）. PDI pro-vides a measure of the narrowness of the particle size dis-

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tribution, with values << 0.1 indicating a very narrow dis-tribution24）.

3 RESULTS AND DISCUSSION3.1 Phase behavior

To investigate how phase behavior changes by replacing water with glycerol in water/mixture of PGPR and PGFA/vegetable oil system, phase diagram of the glycerol/surfac-tant HLP/vegetable oil system were determined.

Figure 1 shows the phase diagram of the water/surfac-

tant HLP/vegetable oil system（A）and the glycerol/surfac-tant HLP/vegetable oil system（B）at 25℃. Figure 1A is constructed based on previous article16） with additional in-vestigations to clearly explain differences between the system with water and the system with glycerol.

For the water/surfactant HLP/vegetable oil system（Fig. 1A）, the two-phase region consisting of Lc＋L3 or lamellar liquid crystal（La）＋L3 was observed at surfactant HLP concentration above 35 wt％. When the concentration of surfactant HLP was less than 35 wt％, a three-phase region consisting of L3＋Wm＋O or Lc＋L3＋O, and a two-phase region consisting of Wm＋O appeared.

Fig. 1　�Phase diagram of (A) water/surfactant HLP/vegetable oil and (B) glycerol/surfactant HLP/vegetable oil systems at 25℃. Phase diagram (A) is constructed based on the previous article16) with additional investigations. L3, Lc, La, Wm, G and O indicate a sponge phase, a liquid crystalline phase, a lamellar phase, a micellar phase, a glycerol phase and an oil phase, respectively. In our previous study, we considered that the position where the concentration of surfactant HLP and water was below 35 wt% and below 20 wt%, respectively (dotted line in Fig. 1A), produced a two-phase region consisting of Lc + L3

16). By additional investigations for the water/surfactant HLP/vegetable oil system, we now presume the existence of a three-phase region consisting of Lc + L3 + O as shown in Fig. 1A.

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Figure 2 shows the polarized microphotographs, and SAXS patterns of mixtures of compositions（a）,（b）and（c）described in Fig. 1B and their visual states between crossed polarizes are indicated in Fig. 3. The sponge phase（L3）is often stated to be flow birefringent25）, and it is re-ported that the SAXS pattern obtained from L3 indicates a broad peak26）. The scattering peak for the sample（a）in Fig. 1B was broad（Fig. 2-a）. On the other hand, this sample showed birefringence under polarization microscopy（Fig. 2-a）and between crossed polarizes in a static state（Fig. 3-a）. Consequently, sample（a）in Fig. 1B was identified as a two-phase coexisting with liquid crystalline（Lc）and L3. As shown in Fig. 2, the SAXS pattern and the polarized mi-crophotograph obtained from sample（b）in Fig. 1B showed a similar result to that of sample（a）. The SAXS pattern of sample（c）indicated a curved shape similar as a skirt of the broad peak of sample（b）shown in Fig. 2. We considered that the SAXS pattern obtained from sample（c）in Fig. 1B was the skirt of broad peak owing to L3（Fig. 2-c）. As shown in Fig. 3-b, c, oil or glycerol separated and the lower layer

of sample（b）or the upper layer of sample（c）showed bire-fringence more intensively in flow state. This behavior is characteristic of L3 phase as described in our previous report16）. These samples showed birefringence under po-larization microscopy（Fig.2-b, c）, and showed birefrin-gence in lower layer for sample（b）and between L3 layer and glycerol layer for sample（c）as shown in Fig. 3. Conse-quently, sample（b）and（c）in Fig. 1B were determined as a three-phase coexisting with Lc, L3 and oil phase（Lc＋L3＋O）, and Lc, L3 and a glycerol phase（Lc＋L3＋G）, respec-tively as shown in Fig. 3.

For the glycerol/surfactant HLP/vegetable oil system（Fig. 1B）, Lc＋L3 was observed in all regions of the phase diagram. The two-phase region（Lc＋L3）appeared at sur-factant HLP concentration above 35 wt％. When surfactant HLP and the vegetable oil concentration was below 35 wt％ and below 20 wt％, respectively, a three phase region with Lc＋L3＋G could be seen. When the vegetable oil con-centration was increased to more than 20 wt％ at surfac-tant HLP concentration less than 35 wt％, vegetable oil was gradually expelled from Lc＋L3, resulting in the forma-tion of a three phase region with Lc＋L3＋O or a four phase region with Lc＋L3＋G＋O. Figure 4 shows a photograph of samples consisting of 40 wt％, 20 wt％ and 40 wt％ of water or glycerol, surfactant HLP, and vegetable oil, re-spectively. The photograph（i）in Fig. 4 represents water/surfactant HLP/vegetable oil system and（ii）in Fig. 4 repre-sents glycerol/surfactant HLP/vegetable oil system. In the system with water, Wm＋O was formed（Fig. 4-i）, whereas in the system with glycerol, Lc＋L3＋O was formed（Fig. 4-ii）. Lc＋L3 could be seen in the glycerol/surfactant HLP/vegetable oil system at the low surfactant content region, but was not observed in this region of the water/surfactant HLP/vegetable oil system. Thus, Lc＋L3 expanded toward lower surfactant concentration when water was replaced with glycerol in the water/surfactant HLP/vegetable oil system.

The expansion of the sponge phase has been observed in previous studies on the ternary system composed of poly-

Fig. 2　�Polarized microscopic images and SAXS patterns of samples consisting of 25.0 wt%, 65.0 wt% and 10.0 wt% (a); 40.0 wt%, 20.0 wt% and 40.0 wt% (b); 70.0 wt%, 20.0 wt% and 10.0 wt% (c) of glycerol, surfactant HLP and vegetable oil, respectively. Compositions (a), (b) and (c) are indicated in Fig. 1B using the corresponding letters in parentheses.

Fig. 3　�Photographs of samples represented by composition (a), (b) and (c) in Fig. 1B between crossed polarizes.

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oxyethylene sorbitan monooleate（MOPS）, 40 w/w％ sugar solution（sucrose, D-fructose, D-glucose, or D-maltose）, and vegetable oil17, 18）. Sugars such as D-glucose, D-maltose, and sucrose have been shown to decrease the cloud point of MOPS27）. A decrease in the cloud point of MOPS by adding sugar therefore suggests that the effective HLB of MOPS shifts toward the hydrophobic side in the presence of sugar. Thus, the expansion of the sponge phase region observed in previous studies has been considered to be caused by increased hydrophobicity of MOPS in the pres-ence of sugar. Glycerol also has been shown to decrease the cloud point of octaethylene glycerol dodecyl ether23）. Therefore, we presume that the expansion of the sponge phase or liquid crystal phase region observed in this study is caused by increased hydrophobicity of PGPR and HGML mixtures in the presence of glycerol. Determining the cloud point of PGPR and HGML mixtures was attempted but the cloud point could not be found for either the water or glycerol systems.

3.2 Preparation of nano-emulsionAn o/w emulsion was prepared as described in the ex-

perimental section（2.2.3）. Figure 5 shows the positions of the mixtures selected to start emulsification in the phase diagram. By overlaying Fig. 5 onto the phase diagrams（Fig. 1A, B）, the phase type of the pre-emulsified mixtures was confirmed.

Table 1 shows the phase type and weight composition of pre-emulsified samples in the water or glycerol/surfactant

HLP/vegetable oil system, and shows the average droplet diameter and PDI of the o/w emulsions prepared from the samples.

Figure 6 is a representative of particle size distribution of o/w emulsions prepared from the samples with different S/O weight ratio in the water/surfactant HLP/vegetable oil system（A）and in the glycerol/surfactant HLP/vegetable oil system（B）.

Figure 7 shows a photograph of the o/w emulsions pre-pared from samples I to VIII indicated in Table 1.

In the case of S/O weight ratio 8:2 in the system with water, when the phase type of pre-emulsified mixtures was Lc＋L3 or L3＋La, the average droplet diameter of the pre-pared o/w emulsions was less than 35 nm and PDI ranged from 0.23 to 0.30（Table 1）. In the case of S/O weight ratios 6:4 and 4:6, o/w nano-emulsions with droplet sizes ranging from 40 to 170 nm could be prepared by addition of water to Lc＋L3 and PDI ranged from 0.15 to 0.37. When the phase type of pre-emulsified mixtures was L3＋Wm＋O, the droplet diameter of the prepared o/w emulsions was more than about 525 nm and PDI ranged from 0.37 to 1.2, and in the case of S/O weight ratio 4:6, the droplet sizes could not be measured.

In the case of S/O weight ratio 8:2 in the system with glycerol, when the phase type of pre-emulsified mixtures was Lc＋L3, the average droplet diameter of the prepared o/w emulsions was less than 30 nm and PDI ranged from 0.27 to 0.32, and the droplet diameter of the prepared o/w emulsions by addition of water to Lc＋L3＋G was less than 35 nm and PDI ranged from 0.28 to 0.30（Table 1）. In the case of S/O weight ratios 6:4 and 4:6, o/w nano-emulsions

Fig. 4　�Photographs of aqueous solution/surfactant HLP/vegetable oil systems under normal light. Aqueous solutions were composed of water (i) and glycerol (ii). Weight ratio of aqueous solution, surfactant HLP, and vegetable oil was 4:2:4 that is a composition (b) indicated in Fig. 1B.

Fig. 5　�The positions of the mixtures selected to start e m u l s i f i c a t i o n i n t h e p h a s e d i a g r a m . Compositions ●, ■ and × indicate S/O weight ratios of 8:2, 6:4 and 4:6, respectively. S/O means weight ration of surfactant to oil.

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with droplet sizes ranging from 45 to 115 nm could be pre-pared by addition of water to Lc＋L3 and PDI ranged from 0.17 to 0.31. When the phase type of pre-emulsified mix-tures was Lc＋L3＋G or Lc＋L3＋G＋O, o/w nano-emulsion or o/w emulsions with droplet sizes ranging from 125 to 545 nm could be prepared and PDI ranged from 0.16 to 0.36, and in the case of the preparing o/w emulsions by ad-dition of water to Lc＋L3＋O, the droplet sizes could not be measured.

In both systems, water and glycerol systems, fine o/w na-no-emulsions could be prepared by addition of water to Lc＋L3, and when oil phase was separated in pre-emulsified samples, fine emulsions could not be prepared. In the case of preparing o/w emulsions by addition of water to Lc＋L3

＋G in the system with glycerol, it was also found that the droplet sizes of emulsions could be relatively small. Replac-ing water with glycerol was responsible for the expansion of the region containing Lc＋L3 toward lower surfactant concentration in the glycerol/surfactant HLP/vegetable oil system; therefore, in the glycerol/surfactant HLP/vegetable

oil system compared with the water/surfactant HLP/vege-table oil system, the region where o/w nano-emulsions or o/w emulsions could be prepared using an isothermal low-energy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller.

Gohtani et al.11） concluded that identifying and clarifying the phase structure existing in the pre-emulsification and determining the relevant phase structure for producing the desired emulsion size during the emulsification process is very important when employing low-energy emulsification methods. In this study, the role of Lc＋L3 in forming o/w nano-emulsions prepared by using an isothermal low-ener-gy method with PGPR and PGFA has been fully described.

As described above, PDI of o/w nano-emulsions prepared in this study was more than 0.1, indicating a relatively wide particle size distribution（Table 1）, and in most cases, par-ticle size distributions showed wide monomodal distribu-tion in S/O weight ratio 8:2 and monomodal or bimodal dis-tribution in S/O weight ratio 6:4 and 4:6（Fig. 6A, B）. The appearance of the emulsions was correlated to their mean

Table 1　�Phase type and weight composition of the pre-emulsified samples in the water or glycerol/surfactant HLP/vegetable oil system and the average droplet diameter and polydispersity index of the prepared o/w emulsions.

S/O weightratio

Weight ratio Water for aqueous solution Glycerol for aqueous solutionSamplenumberHLP Oil

Aqueoussolution

Phase typeAverage dropletdiameter (nm)

Polydispersityindex

Phase typeAverage dropletdiameter (nm)

Polydispersityindex

8 : 2

7.62 1.90 0.48 Lc+L3 25.2 0.242 Lc+L3 28.1 0.271 I

7.27 1.82 0.91 Lc+L3 24.2 0.262 Lc+L3 26.5 0.265 II

6.67 1.67 1.67 Lc+L3 34.9 0.231 Lc+L3 23.3 0.290 III

5.00 1.25 3.75 Lc+L3 24.6 0.293 Lc+L3 17.4 0.292 IV

4.00 1.00 5.00 L3+La 18.6 0.303 Lc+L3 15.1 0.312 V

3.20 0.80 6.00 Lc+L3 14.7 0.283 Lc+L3 17.2 0.318 VI

2.00 0.50 7.50 L3+Wm+O 2428.5 1.165 Lc+L3+G 29.7 0.278 VII

1.00 0.25 8.75 Wm+O UM* － Lc+L3+G 35.5 0.299 VIII

6 : 4

5.71 3.81 0.48 Lc+L3 60.6 0.294 Lc+L3 46.2 0.312 I

5.45 3.64 0.91 Lc+L3 56.4 0.296 Lc+L3 47.7 0.309 II

5.00 3.33 1.67 Lc+L3 82.3 0.268 Lc+L3 49.7 0.297 III

3.75 2.5 3.75 Lc+L3 41.6 0.326 Lc+L3 53.2 0.271 IV

3.00 2.00 5.00 L3+Wm+O 526.3 0.369 Lc+L3+G 307.4 0.362 V

2.40 1.60 6.00 Wm+O 537.3 0.289 Lc+L3+G 354.7 0.300 VI

1.50 1.00 7.50 Wm+O UM* － Lc+L3+G 354.8 0.275 VII

0.75 0.50 8.75 Wm+O UM* － Lc+L3+G 126.3 0.257 VIII

4 : 6

3.81 5.71 0.48 Lc+L3 122.5 0.152 Lc+L3 114.7 0.186 I

3.64 5.45 0.91 Lc+L3 164.5 0.207 Lc+L3 112.2 0.171 II

3.33 5.00 1.67 Lc+L3 144.1 0.186 Lc+L3+O UM* － III

2.50 3.75 3.75 Wm+O UM* － Lc+L3+O UM* － IV

2.00 3.00 5.00 Wm+O UM* － Lc+L3+O UM* － V

1.60 2.40 6.00 Wm+O UM* － Lc+L3+G+O 542.7 0.215 VI

1.00 1.50 7.50 Wm+O UM* － Lc+L3+G 541.4 0.250 VII

0.50 0.75 8.75 Wm+O UM* － Lc+L3+G 217.1 0.155 VIII

UM：unmeasurable level

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particle diameters rather than particle size distributions. The o/w nano-emulsions prepared in S/O weight ratio 8:2 and 6:4 were transparent（Fig. 7A I–VI, B I–VIII）and bluish translucent（Fig. 7C I–IV, D I–IV）, respectively, in spite of their wide particle size distribution. In S/O weight ratio 4:6, the appearance of formed o/w nano-emulsions or emulsions were milky white（Fig. 7E, F）; nevertheless their PDI was relatively low and particle size distributions were slightly narrower than those for the o/w emulsions prepared in S/O weight ratio 8:2 and 6:4（Table 1, Fig. 6A, B）.

4 CONCLUSIONWe have investigated how phase behavior changes by re-

placing water with glycerol in water/mixture of PGPR and HGML（surfactant HLP）/vegetable oil systems, and deter-mined the effect of incorporating glycerol in mixtures of PGPR and HGML on o/w nano-emulsion formation using an

isothermal low-energy method. In the phase behavior study, a two-phase region with coexisting liquid crystalline（Lc）and sponge phase（L3）expanded toward lower surfac-tant concentration when water was replaced with glycerol in a water/surfactant HLP/vegetable oil system. The droplet sizes of the emulsions prepared from a mixture of PGPR and HGML with water or glycerol was examined and it was found that o/w nano-emulsions were formed by emulsifica-tion of samples in a region containing Lc＋L3. These results indicate that Lc＋L3 is an important factor in preparation of o/w nano-emulsions using an isothermal low-energy method with PGPR and PGFA. The PDI of o/w nano-emul-sion prepared in this study indicated a relatively wide par-ticle size distribution（more than 0.1）, however, the appear-ance of the emulsions could be correlated with their mean particle diameters rather than with their particle size dis-tribution. When weight ratio of surfactant to oil was higher, prepared o/w nano-emulsions had a relatively wide particle size distribution, but their average droplet sizes and ap-pearance were smaller and transparent or bluish translu-cent, respectively. In the case of lower weight ratio of sur-factant to oil, the appearance of prepared o/w nano-emulsions or emulsions were milky white, however, their PDI was slightly less and particle size distribution were slightly narrower. Additionally, in the glycerol/surfactant HLP/vegetable oil system, replacing water with glycerol was responsible for the expansion of the region containing Lc＋L3 toward lower surfactant concentration, and as a result, in the glycerol/surfactant HLP/vegetable oil system compared with the water/surfactant HLP/vegetable oil system, the region where o/w nano-emulsions or o/w emul-sions could be prepared using an isothermal low-energy emulsification method was wide, and the droplet diameter of the prepared o/w emulsions was also smaller. Therefore, glycerol was confirmed to facilitate the preparation of na-no-emulsions from a system containing surfactant HLP. Moreover, in this study, we could prepare o/w nano-emul-sions with a simple one-step addition of water at room temperature without using a stirrer. Thus, the present technique is highly valuable for applications in several in-dustries.

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Fig. 6　�Particle size distribution of o/w emulsions prepared from the samples with different S/O weight ratio in aqueous solution/surfactant HLP/vegetable oil system. Aqueous solutions were composed of water (A) and glycerol (B). The selected samples were the number (II) for each S/O weight ratio noted in Table 1.

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Fig. 7　�Photographs of o/w emulsions prepared using the method described in 2.2.3. (A), (C) and (E) indicate the o/w emulsions prepared from S/O weight ratio of 8:2, 6:4 and 4:6, respectively, in the system with water. (B), (D) and (F) shows the o/w emulsions prepared from S/O weight ratio of 8:2, 6:4 and 4:6, respectively, in the system with glycerol. The sample number (I–VIII) refers to the number noted in Table 1.

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