Preparation and stabilization of simple and multiple emulsions using a microporous glass membrane

COLLOIDS AND SURFACES ELSEVIER

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Colloids and Surfaces B: Biointerfaces 6 (1996) 261-268

Preparation and stabilization of simple and multiple emulsions using a microporous glass membrane Yoshinori Mine a,,, M a s a a k i Shimizu b, Tadao N a k a s h i m a b a Department of Food Science, University of Guelph, Guelph, Ontario, NIG 2W1, Canada b Industrial Research Institute of Miyazaki Prefecture, Tsunehisa, Miyazaki 880, Japan Received 29 June 1995; accepted 12 December 1995

Abstract

A microporous glass membrane with a narrow range of pore sizes was used for the preparation of simple and wateroil-water (W/O/W) type emulsions using egg yolk phospholipids and soybean oil. Simple oil-in-water (O/W) and water-in-oil (W/O) emulsions and a W/O/W type emulsion were successfully prepared using the membranes without any coalescence of oil drops or breakdown of the emulsions. The simple and W/O/W emulsions were stable for at least 6 weeks when stored at 5°C. The particle size distribution of the emulsion depended on the pore size of the membrane. These results indicate that the technique would be valuable for the production of stable simple and multiple emulsions for food uses as well as for intravenous fat and/or drug carriers. Keywords: Emulsion stability; Egg yolk Phospholipids; Microporous glass membrane; Multiple emulsion; Oil-in-water emulsions; Soybean oil; Water-in-oil emulsions

I. Introduction

Both oil-in-water (O/W) and water-in-oil (W/O) emulsion systems are often prepared as foods, pharmaceuticals and cosmetics. It is well known that, since the emulsion is thermodynamically unstable, flocculation and coalescence immediately occur after emulsification. Generally, the stability of an emulsion greatly depends upon the emulsifying agent, droplet size, net charge and mechanical and physical properties of the adsorbed film [1]. In particular, the distribution of emulsion droplets is the most important parameter in characterizing any emulsion. Stability and resistance to creaming, rheology, chemical reactivity and physiological efficiency are influenced by both the relative size * Corresponding author. 0927-7765/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0927-7765(95)01264-8

and the size distribution [2]. O p t i m u m particle design and its strict size control are very important factors not only for the preparation of stable emulsions but also for discovering novel dispersions which contain solid or liquid particles. Various instruments exist both on the industrial and laboratory scale to produce emulsions, e.g. colloid mills, toothed discs, dispersing machines, and high-pressure homogenizers [2,3]. The emulsions made by these instruments show considerable polydispersity such that the droplet size distribution is usually between 0.1 and 100 gin. In general, polyphase emulsions are thermodynamically unstable and tend to separate unless stabilized. The preparation of simple emulsions is considered to be more difficult using the above instruments. A new emulsifying technique, called membrane emulsification, was proposed by N a k a s h i m a et al. [4].

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In this technique, a microporous glass membrane with a narrow pore size distribution was used as an emulsifying tool to establish a convenient and more useful technology in the design of an emulsion. The membrane emulsification method is based on the principle of dispersing one of two immiscible liquids (the dispersion phase) into the other (the continuous phase) by applying pressure to cause the dispersion phase to permeate through the membrane [5]. This method has the capability of producing not only simple emulsions, but also multiple emulsions such as W/O/W type emulsions. However, Nakashima et al. mainly studied the kerosene/water emulsion system for preparing simple emulsions [4]. Only a few studies on the preparation of emulsions using triglyceride have been reported [6]. Phospholipids (PLs) occur widely in nature (e.g. soybean and egg yolk) and are an important component of biomembranes. Phosphatidylcholine (PC) is a common PL, is important as a naturally occurring emulsifier and is widely used as a stabilizing agent in food emulsions [7]. Lysophosphatidylcholine (LPC) exists as a minor component, but has diverse functions such as interaction with PC and the formation of a stable emulsion [-8,9]. Natural emulsifiers such as PLs are preferable to artificial emulsifiers and have proven to be very useful in the preparation of intravenous fat and/or drug carrier emulsions [ 10,11 ]. Regulation of the particle size of emulsions made from PLs is required in order to define their applications in cosmetic or medical uses. A W/O/W multiple emulsion is an O/W emulsion in which the dispersed oil drops themselves contain smaller dispersed aqueous droplets. Therefore, the oil layer between the two aqueous phases (internal and external) can act as a membrane. Recently, there has been increased interest in the uses of W/O/W emulsions in many fields, such as microencapsulation [12], immobilization of enzymes and detoxification of blood [ 13], and prolongation of drug release [14-16]. Although these emulsions have shown potential applications in such areas, the preparation of multiple-phase emulsions has been limited because of their complex nature. A reliable method for preparing W/O/W emulsions has not yet been established. If

it were possible to prepare specified stable simple or multiple emulsions by natural emulsifiers such as PLs, the utilization of emulsions could be expanded. In the present work, simple and W/O/W type multiple emulsions composed of soybean oil and egg yolk PLs were prepared using the microporous glass membrane method. The stability of these emulsions was also investigated.

2. Materials and methods

2.1. Materials Soybean oil was purchased from Sigma Chemicals, St. Louis, M.O. Egg yolk PC and LPC, 98% pure, were kindly provided from Q.P. Corporation, Tokyo, Japan and were used without further purification. Polyglycerol esters of polycondensed ricinoleic acid (PGCR) were obtained from Sakamoto Yakuhin Co. Ltd., Osaka, Japan. Other chemicals were obtained from Sigma. Microporous glass membranes made with A 1 2 0 3 - S i O 2 type glasses (hydrophilic membrane) were purchased from Ise Chemical Corp., Tokyo, Japan, and were 0.36-1.361xm in diameter, 0.5ktm thick and 230 mm in length. O / W emulsions were prepared using a hydrophilic membrane immersed in 2 N HC1 solution for 2 h and then rinsed in distilled water. A hydrophobic membrane was used to prepare W/O emulsions according to the method of Nakashima et al. [5]. A hydrophilic microporous membrane was heated at 200°C for 48 h in vacuo. It was then immersed in toluene to which 5% (v/v) octadecyltrichlorosilane (ODS) was added, and the mixture was refluxed at 110°C for 8 h. The toluene was previously dried by dehydration with anhydrous zeolite 4A. The ODS-treated microporous membrane was then treated at room temperature for 2 h in dry toluene to which 1% (v/v) trimethylchlorosilane (TMS) was added. Finally, the membrane was rinsed in dry toluene. 2.2. Apparatus Fig. 1 shows a schematic diagram of the membrane emulsifying process, and the apparatus used

Y. Mine et al./Colloids Surfaces B: Biointerfaces 6 (1996) 261-268

263

loaded into the soybean oil phase (containing 0.5% PC and PGCR) through the membrane.

2.4. Preparation of W/O/Wmultiple emulsion

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Fig. 1. Schematic diagram of the membrane emulsification process and the experimental apparatus.

in this study. This system consists of the membrane module (a), a nitrogen gas tank (b) as a pressure source, the dispersion phase storage tank (c), the emulsion tank (d) and the circulation pump (e). The protocol for monodispersed emulsion formation is also shown in this figure. The microporous membrane was prewetted with the continuous phase, prior to the start of the membrane emulsification, i.e. the hydrophilic membranes for O / W emulsions were fully wetted with the water phase and the hydrophobic membranes for W/O emulsions were fully wetted with the soybean oil phase. Wetting of the membranes was effectively conducted by immersing the microporous membrane in the continuous phase and degassing it under vacuum, while subjecting it to ultrasound for 30 rain. The membrane was then carefully attached to the membrane module.

2.3. Preparation of simple O/Wand W/O emulsions Simple O / W and W/O emulsions were prepared as follows. After a hydrophilic microporous membrane was inserted into the module of the emulsification apparatus, soybean oil in the storage tank was loaded into the water phase (containing 5.0% glucose or 1.0% NaC1) through the membrane. L P C was dispersed in the water phase, while PC was dispersed in the oil phase. The water phase was circulated during this process by pumping. In the case of W/O emulsions, a hydrophobic microporous membrane was inserted into the module and the water phase (containing 5.0% glucose) was

A W/O/W emulsion is representative of a multiple emulsion. First, the W/O emulsion with a 30% (v/v) water concentration (composed of 0.5% PC and PGCR) was prepared using a Microfluidizer (model 110S, Microfluidics Corp., Newton, MA) at an input pressure of 0.3 MPa, which corresponds to a pressure drop of 42 M P a during the homogenization procedure. The sample was circulated through the homogenization chamber ten times to ensure minimum particle size. The mean diameter of the W/O emulsion was 0.54 rtm. This W/O emulsion was then loaded into the water phase (containing 1.0% LPC and 5.0% glucose) through a hydrophilic microporous membrane. W/O/W emulsions formed by osmotic pressure were prepared as follows. A W/O emulsion with 30% water and 30% glucose was obtained by the method described above. The mean droplet size of the emulsion was 0.52 ~tm. The emulsion was then diluted with soybean oil to obtain 0.1% water droplets and 30% glucose, and the W/O emulsion was loaded into the water phase containing 1.0% LPC and 1.0% glucose through a hydrophilic microporous membrane. Changes of particle size relative to osmotic pressure were followed by means of a laser-diffraction-type particle size analyzer and confirmed by use of an optical microscope.

2.5. Particle size analysis The size distribution of the emulsion droplets was measured by means of a laser-diffraction particle analyzer (model SALD-2000, Shimadzu Corp., Kyoto, Japan). This particle analyzer was also used to assess the stability of the emulsions during aging.

2.6. Water concentration analysis The concentration of the water in the oil phase was measured according to the Karl-Fisher method [17] using a Karl-Fisher moisture meter (model

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Y. Mine et al./Colloids Surfaces B: Biointerfaces 6 (1996) 2 6 1 ~ 6 8

KAF-1, Tsutsuki Rikagaku Kiki Co., Ltd, Tokyo, Japan).

3. Results and discussion

3.1. Preparation of simple O/Wand I41/0 emulsions Typical micrographs and droplet size distributions of the O/W and W/O emulsions composed of soybean oil and egg PLs prepared using hydrophilic membranes (average pore size, Dm= 1.36 ~tm and 0.36 tam) and hydrophobic membranes (D m = 1.00 ~tm) are shown in Fig. 2, and the properties of the emulsions are summarized in Table 1. Both types of emulsion were successfully prepared with a uniform particle size by the membrane emulsification method. The size distributions of the emulsions were very narrow. The mean diameter of simple O / W emulsions was 3.38 ~tm ( D m = 1 . 3 6 ) and 1.07 pm (Din = 0.36 ktm), and over 90% of the total volume of the oils were incorporated into emulsions with droplet sizes from 2.06 to 5.02 pm (Dm=l.36 Ixm), and from 0.81 to 2.90 pm (Dm = 0.36 ~tm), respectively. The simple W/O emulsion exhibited a mean diameter of 3.08 lam and over 90% of the total volume of oil was in the form of particles of diameters between 1.67 and 6.02 ktm. The distribution of the W/O emulsion was a little wider than the distributions of the simple O/W emulsions. This seems to be due to the heterogeneity of the modified hydrophobic membrane used. The surface areas of the emulsions increased with a decrease of the droplet sizes of the emulsions. The droplet size of the emulsion depended on the D m value of the membrane used. Stable W/O emulsions were not obtained using egg PC alone as a surfactant; however, a fine emulsion with a sharp droplet size distribution was obtained when PC was combined with PGCR. When PC was dispersed in the water phase, a simple O/W emulsion could not be obtained due to the clogging of the membrane with PC micelles. It was reported that when a membrane surface had a charge opposite to that of the functional group in the surfactant molecule, adsorption of the surfactant on the membrane occurred and a simple emulsion could not be formed [4]. When a micro-

porous glass membrane is used without any modification, it exhibits a strongly hydrophilic nature due to the many silanol groups that are present on the glass surface. Hence, the extent of charge on the surfactants in solution can affect the production of simple emulsions. Sodium dodecyl sulfate, which consists of amphipolar molecules that have hydrophobic as well as ionized zones, has often been used for membrane emulsification in order to avoid interactions with the membrane surface. In the present study, triglyceride/egg PL emulsions, considered to be difficult to prepare, were successfully prepared by membrane emulsification. LPC was a more suitable surfactant than PC for membrane emulsification because of the high ionic charge of the headgroup of the LPC molecule. Emulsion systems comprising suitable oils emulsified using PL materials also have an important role in medical practice. Parenteral emulsions can be used not only for drug administration, but also for the delivery of vaccines, as diagnostic agents, and for nutritional purposes 1-18]. The ability to control the particle size of intravenous fat emulsions and/or drug carrier emulsions should have practical implications.

3.2. Preparation of W/O/Wmultiple emulsions Fig. 3 shows photomicrographs of two types of W/O/W multiple emulsions composed of egg PLs and soybean oil. The W/O emulsion with a mean diameter of 0.52 lxm successfully penetrated into the O / W emulsion, and a high yield of double emulsion was obtained easily using another hydrophilic membrane (Din = 1.0 txm). The concentration of the inner W/O emulsion was about 30%, and the mean droplet size of the outer O / W emulsion was 4.8 ~tm (Fig. 3a). The size of the inner W/O emulsion increased with increasing difference in the osmotic pressure between the inner water droplets (30% glucose) and the outer continuous water phase (1% glucose) (Fig. 3b). No coalescence of water droplets occurred in this process. The water concentration of the inner W/O emulsion was about 30%. Neither of the W/O/W multiple emulsions changed when stored at 5°C for 6 weeks (data not shown). In the preparation of a stable W/O/W multiple

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Table 1 Characterization of simple O / W and W/O emulsions derived from the microporous glass membranes Emulsion type

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a Emulsion distribution range of over 90% of total oils. The data represent the m e a n value of duplicate measurements.

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Fig. 3. Photomicrographs of W/O/W multiple emulsions prepared using porous glass membranes. (a) A W/O/W emulsion prepared using the membrane; (b) a W/O/W emulsion prepared using the membrane and allowed to have a osmotic pressure slope due to the addition of glucose.

emulsion, the selection of the pore size of the m e m b r a n e and the water droplet concentration of the inner W/O emulsion is very important. In general, a hydrophilic membrane for the second step which has a pore size greater than or equal to twice the diameter of the water in the first step W/O emulsion is required. If the pore size of the second membrane is equal to or smaller than the diameter of the water particles, the water particles will be rejected by the membrane so that it will be impossible to obtain a double emulsion. The concentration of water droplets for W / O / W emulsions should be between 30 and 50% in order to obtain a stable double emulsion [5]. It is expected that a W / O / W emulsion would be suitable for the development of slow-release drug delivery systems or in selectively transferring a drug into the lymphatic system [ 19]. However, there have been few applications of this type of emulsion as a drug carrier via the enteral route because of the lack of a reliable method for preparing multiple emulsions. An attempt to prepare a W / O / W multiple phase emulsion using an artificial nonionic surfactant was reported [ 15,20]. For pharmaceutical or cosmetic applications, the safety of the emulsion products, the toxicity and the effects of the emulsifier on the body must be taken into consideration [18]. In light of this, natural emulsifiers such as soybean or egg yolk PLs are preferable to artificial emulsifiers. In the present study, membrane emulsi-

fication was found to be a viable technique for the preparation of simple or multiple emulsions (soybean oil and egg PLs) which could be used for food, cosmetic, and medical applications. However further studies are required to prepare inner W/O emulsions using only natural surfactants for safety reasons. 3.3. Stability o f simple O/Wemulsions

Fig. 4 shows changes in a simple O / W emulsion prepared using a membrane (Dm--=1.36 lam), and in a polydispersed emulsion prepared with a disperser composed of 1.0% egg PC and 20% soybean oil after storage for 6 weeks at 5 °C. The size distribution of the simple emulsion (mean diameter, 3.32 ~tm) was not affected by aging. Slight creaming was observed, but no coalescence or breakdown of droplets was seen during storage. The emulsion exhibited a sharp droplet size distribution from 1.39 to 4.50 ~tm (mean diameter, 3.38 ~tm). On the other hand, the emulsion prepared with the disperser exhibited a wide and heterogeneous droplet size distribution from 0.32 to 41.89 lam, which tended to larger droplet sizes during aging due to coalescence and breakdown. The mean droplet size changed from 4.37 to 15.56 ~tm, and the droplet size distribution ranged from 0.5 to 256.4 ~tm. The stabilization of emulsions is important, with applications in the chemical industry, in the formulation

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of drugs, and in food production. The stability of emulsions is influenced by various factors, e.g. viscosity, energy barriers and van der Waals interactions [ 3 ]. These results indicate that the dispersity of the emulsion droplets is an important parameter in stabilizing PL emulsions as well as droplet sizes.

3.4. Relationship between D m and emulsion droplet size As described previously, the droplet size depended on the D m value of the membrane used to prepare the emulsion. This raises the possibility of controlling the emulsion droplet size using various pore size membranes under the condition that a relationship exists between Dm and the emulsion droplet size. The relationship between the Dm value of the membranes and the mean droplet size of the emulsions formed by these membranes is shown in

Fig. 5. The particle size distribution of the emulsion was directly related to the pore size distribution of the membrane used and showed the desired simple dispersity. It was found that the mean diameter (Dp) of the egg P L emulsions was directly proportional to Dm value and was determined by use of the equation Dp=3.18 Dm+0.42. This indicates that microporous glass membranes allow for the free exchange of droplets in the submicron to micron range, thus allowing the customized design of ideal emulsions.

Acknowledgment The authors thank Professor R.Y. Yada at the Department of Food Science, University of Guelph, for his critical reading of the manuscript.

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