Polysaccharide gel with multiple emulsion

Food Hydrocolloids 19 (2005) 605–615 www.elsevier.com/locate/foodhyd

Polysaccharide gel with multiple emulsion Julia Weiss*, Inta Scherze, Gerald Muschiolik Department of Food Technology, Institute of Nutrition, Friedrich-Schiller-University Jena, Am Steiger 3, D-07743 Jena, Germany

Abstract The aim of the present work was to investigate the possibility of using a semicrystalline oil phase in W/O/W to modify the release of encapsulated hydrophilic compounds from polysaccharide gels with embedded multiple emulsions. L-Tryptophan was enclosed within the W1-phase of an W1/O/W2-emulsion, which itself was homogeneously distributed in a Ca2C-alginate gel with maltodextrin (D.E. 6.5) as a bulking agent. Various lipid phases, including MCT-oil and different vegetable fats, were investigated for their ability to act as a hydrophobic barrier in the oil phase. In addition, the content of the lipophilic surfactant PGPR in the oil phase was varied. A marked increase in encapsulation efficacy was observed by lowering the storage temperature of the emulsion gels from 23 to 7 8C. Also, among the various lipids used for sample preparation, differences in release rate were found. Increasing the fat content in the oil phase resulted in decreased marker diffusion. The lowest release rate was achieved by using the fat with the highest melting point and by increasing the fat content to 100% of the oil phase. Lowering the content of PGPR in the MCT-oil phase (without fat) had no effect on the release of tryptophan. q 2004 Elsevier Ltd. All rights reserved. Keywords: Alginate emulsion gels; W/O/W emulsion; Effect lipid phase composition; Release

1. Introduction The entrapment of water-soluble low molecular weight substances (LMWS) is often very difficult due to their rapid leakage out of various encapsulation systems, especially when food-grade ingredients are used to prepare the capsules (Schrooyen, van der Mer, & De Kruif, 2001). Typical matrices that are often used to entrap water-soluble ingredients include polysaccharide gels such as cross-linked alginates or pectin, or multiple emulsions, especially the W/O/W-type emulsion. Due to increasing knowledge of the stabilization of multiple emulsions, these kinds of emulsions represent a very promising encapsulation method for both hydrophilic and lipophilic compounds (Auweter, 2001). Limitations, however, exist in the use of multiple emulsions as encapsulation matrices since a mechanically solid and rigid water-insoluble capsule is required to enclose a sensitive drug. For this reason, several drying processes are widely used and different matrix-forming polymers with

* Corresponding author. Tel.: C49 3641 949716. E-mail address: [email protected] (J. Weiss). 0268-005X/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foohyd.2004.10.023

special properties are available. Such polymers are used to increase the viscosity of the system and to generate a solid matrix that is resistant to mechanically-induced strain. One of them is the polysaccharide alginate, which forms gels in the presence of cross-linking calcium ions (Moe, Draget, Skja˚k-Bræk, & Smidsrød, 1995). Alginate represents a major entrapment system for cells and higher molecular substances such as enzymes and proteins (Murano, 1998; Poncelet & Markvicheva, 2001). Depending on the source of the alginate and molecular composition these gels have pores ranging from 5 to 200 nm in diameter, which determine the entrapment efficacy (Smidsrød & Skjak-Braek, 1990; Stewart & Swaisgood, 1993; Li, Alttreuter & Gentile, 1996). It has been shown that the diffusion of small hydrophilic molecules occurs undiminished throughout the gel matrix (Kikuchi, Kawanabe, Sugihara, Sakurai, & Okano, 1999; Li et al., 1996; Tanaka, Matsumura, & Veliky, 1984). Combining different encapsulation techniques offers the possibility of creating a new encapsulation system with adjustable release properties and additional protection against drug leakage and drug destruction mediated by environmental conditions. Thus, it is possible to embed

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a multiple emulsion within an alginate gel matrix where the gel phase represents the outer phase of the multiple emulsion. Nakhare and Vyas (1995) prepared a W/O/gel system where the outer gel phase consisted of 5% polyacrylic acid, a gelling polymer. They encapsulated a water-soluble substance within the inner aqueous W1-phase and observed a prolonged release of this compound. Furthermore, stable multiple emulsions were formulated ¨ zer, Muguet, Roy, Grossiord, and Seiller (2000) using by O different viscosifying and gelling agents in the outer aqueous phase. The compactability, thickness, and stability of these emulsions increased at room temperature as well as at 40 8C. A desired increase in the release of NaCl with shear rate was observed in multiple emulsions containing different gelling polymers in the outer aqueous phase as compared to systems without the addition of a polymer (Muguet et al., 2001; Olivieri et al., 2001). The transport of water and LMWS in multiple emulsions of the W/O/W-type occurs by diffusion across the oil layer from one water phase to the other (Jager-Lezer et al, 1997; Laugel, Chaminade, Baillet, Seiller, & Ferrier, 1996; Pays, Giermanska-Kahn, Pouligny, Bibette, & Leal-Calderon, 2002) or by formation of micelles composed of the surfactant and the oil (Hino, Shimabayashi, Tanaka, Nakano, & Okochi, 2001). By adding semicrystalline phases or lipids with a high melting point to the oil phase, the viscosity of the oil phase would be expected to increase. Westesen, Drechsler, and Bunjes (2000) postulated a higher release of active substances from O/W emulsions prepared with high melting lipids, when the emulsion droplets were liquid. Garti, Aserin, Tiunova, and Binyamin (1999) formulated a W/O/W emulsion with microcrystalline fat particles of submicron size in the oil phase. The adsorption of these particles to the W/O interface led to an effective stabilization of the system and besides this no release of the encapsulated marker (NaCl) occurred. It is well known that thick layers of the capsule shell consisting of narrow polymer meshes combined with immiscible phases (e.g. W/O/W or O/W/O) and/or enclosed crystalline phases can

suppress diffusion and release of LMWS (Pothakamury & Barbosa-Canovars, 1995). The objective of the present work was to examine the possibility of using a semicrystalline oil phase in W/O/W to modify the release of encapsulated hydrophilic compounds. For this study, polysaccharide gels with embedded multiple emulsions of the W/O/W-type containing the bioactive substance were prepared. The LMWS L-tryptophan was enclosed within the inner aqueous phase of the W/O/Wemulsion, which itself was homogeneously distributed in a Ca2C-alginate gel containing maltodextrin as a bulking agent. Different emulsion lipid phases, i.e. MCT-oil and various vegetable fats, were investigated under the same heating and cooling conditions for their ability to act as a hydrophobic barrier in the emulsion. In addition the content of fat in the oil phase was varied by preparing blends of selected fats in MCT-oil and incorporating them at 50 8C to form the double emulsion. In order to initiate the release of the marker, the encapsulation system W/O/gel was destabilized by storing the gel in water, creating a high osmotic gradient. With these experiments, information about the ability of each individual lipid to influence the mass transport of hydrophilic agents out of the emulsion gel was obtained. L-Tryptophan, with a molecular weight which is small compared to other potential markers, was chosen to assess the encapsulation ability of different lipids because it diffuses more rapidly than larger molecules. 2. Materials and methods 2.1. Materials Descriptions of the oil and fats used as the hydrophobic phase in multiple emulsion preparation are summarized in Table 1. The low HLB surfactant PGPR (IMWITOR 600w) was obtained by SASOL Germany GmbH. The hydrophilic surface-active agent whey protein isolate (WPI, Development Sample DSE 5669, 97.4% protein in dry weight) was

Table 1 Characteristics of the different lipid phases tested for preparing the multiple emulsions Lipid phase a

MCT-oil (MCT) Witocan (Wit)a

Chocosine (Choc)b Crokcool (Crok)c Hardstock (Hard)b

Specification

SFC

Caprylic/capric triglycerides Hydrogenated coco-glycerides, saturated even numbered and non-branched plant fatty acids with chain numbers C10–C18, TM 42–44 8C Cacao butter equivalent (CBE) fat, 34.1% C16, 29.1% C18:1, 31.9% C18:2, 3.1% C18:2, 1.8% others, TM 33–35 8C Fractionated non-hydrogenated Laurin-free refined vegetable fat, 64% saturated fatty acids, 32% mono-unsaturated fatty acids, 4% poly-unsaturated fatty acids, TM 37 8C Reesterified coco-oil and palm stearin, 9.4% C12, 3.4% C14, 43.5% C16, 4.3% C18:1, 7. 2% C18:2, TM 32–35 8C

10 8C: 84% 20 8C: 78% 25 8C: 68% 20 8C: 84% 25 8C: 73%

TM, melting point; SCF, solid fat content (data from producers). a SASOL Germany GmbH. b WALTER RAU Neusser Oil and Fat AG (Germany). c Loders Croklaan B.V. (Netherlands).

10 8C: 75% 20 8C: 56% 25 8C: 44%

J. Weiss et al. / Food Hydrocolloids 19 (2005) 605–615

provided by Fonterra (Europe) GmbH (Germany). The gelling agent gelatine (Bloom 180) was obtained from Gelita Deutschland GmbH and maltodextrin with D.E. 6.5 was supplied by Cerestar (Germany). Sodium alginate (GRINDSTEDw Alginate FD 150 containing 60% mannuronic acid and 40% guluronic acid) was provided by Danisco Cultor (Germany). L-Tryptophan, trichloroacetic acid (TCA), glycerine (Roth, Germany), sodium chloride, CaSO4$2 H2O, Na(PO3)n (Merck, Germany), and the bactericide NaN3 (Merck-Schuchardt, Germany) were of analytical grade (O99 and R86% for glycerine). All experiments were performed using deionised water (!5 mS cmK1). 2.2. Preparation of multiple emulsions The multiple emulsions were prepared using a two-step emulsification procedure. First, the primary water-in-oilemulsions (W1/OZ20/80) were prepared by dispersing the water phase containing NaCl (0.1 M), gelatine (5%) and L-tryptophan (0.05 M) in the oil phase containing PGPR (2 or 8%) with a rotor-stator-system (20,500 rpm for 2 min after addition of the water phase with a velocity of 15 ml minK1; CAT X620, Zipperer GmbH, Germany). Before starting the emulsification process both phases were heated to 60 8C. The freshly prepared W/O was stored at 7 8C for 40 min. This primary emulsion was then gradually dispersed into a WPI solution (1.0%) under moderate stirring (1500 rpm) at 20 8C (emulsions with MCT-oil) or at 55 8C (emulsions with fat), in order to obtain the premix (40 vol% primary emulsion). The final W/O/W emulsion was obtained by homogenizing the premixed emulsion using a low pressure laboratory-scale homogeniser with two orifice valves (operating pressure 1 MPa) (Muschiolik, Roeder, & Lengfeld, 1995). When using a fat as the lipid phase the preparation of the fine emulsion was also carried out at 55 8C. The storage conditions of all multiple emulsions with a fat phase were as follows: emulsions were allowed to cool until ambient temperature was reached (23 8C) and then stored overnight in the refrigerator (7 8C). 2.3. Measurement of droplet size of W/O and W/O/W Droplet size distributions and average droplet sizes (d32 and d43) were determined immediately after preparation using a laser diffraction particle analyser Coulter LS 100 with a microvolume module cell (Coulter Electronics, USA). Droplet size measurements of W/O and the O-phase (W/O/W) were performed in mineral oil (Velocite No.3, Carl Herzog oG, Germany) and disnised water, respectively. 2.4. Release of tryptophan during emulsification and storage The multiple emulsions were characterized by measuring the concentration of tryptophan leaked from W1 to W2

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during the second stage of emulsion preparation and after a storage period of 24 h. The experimental procedure was as follows (Dickinson, Evison, Owusu, & Williams, 1994): Two samples of the freshly prepared multiple emulsion (3 g) were placed in separate centrifuge tubes. One of them was diluted with 2 g of pure water (sample A), the other one with 2 g of a known quantity of a 0.74!10K5 M tryptophan solution (sample B). Both tubes were centrifuged at 1.25!104g for 30 min. An aliquot of the aqueous layer was treated with an equivalent volume of trichloroacetic acid in order to precipitate residues of WPI in the outer aqueous water phase. This solution was filtered, diluted if necessary and the absorbance was recorded at a fixed wavelength (280 nm). The amount (%) of the marker in the outer phase was m0 Z ðcA mB Þ=ðcB K cA Þ, where mB is the amount of tryptophan added to sample B and cA and cB are the photometrically determined concentrations of the marker derived from samples A and B, respectively. The percentage release of tryptophan during homogenisation and after storage is then Release ð%ÞZ 100 ! ½1K ðmi K m0 Þ=mi  where mi is the original theoretical amount of tryptophan encapsulated within the internal droplets. This experiment was done in triplicate for each multiple emulsion. 2.5. Microscopic observation The emulsions were observed by means of a light microscope (Olympus BX61) with ColorView camera and analySISw software (Soft Imaging System). Images of the alginate matrices were made by scanning electron microscopy (JSM 820, JEOL, Japan). A sample of the gel (2 ml) was first frozen with liquid nitrogen. It was then inserted into a cryo-preparation apparatus where it was fractured. Gold was used for sputter-coating the surface of the sample. The prepared sample was then inserted into the scanning electron microscope under vacuum at K185 8C. 2.6. Preparation of alginate gels with embedded multiple emulsion (emulsion gels) and study of release In order to prepare the alginate solutions the dry ingredients (alginate and maltodextrin D.E. 6.5) were mixed together prior to the addition of the necessary amount of deionised water. These solutions were mechanically stirred for at least 3 h at room temperature and then allowed to soak overnight. Using this procedure the occurrence of air bubbles within the viscous solution was markedly reduced. Immediately after its preparation, the multiple emulsion was mixed with the alginate solution. The objective was to obtain emulsion gels with a volume fraction of multiple oil globules of 25% (this value corresponds to 20% oil or fat within the gel matrix). Emulsion gels with MCT-oil were prepared at 20 8C, each containing a fat at 30 8C in order to achieve a homogeneous distribution of the fat droplets in the matrix. For this reason the alginate solution was also

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heated to 30 8C. In the final emulsion gel the concentrations of alginate and the filler were 1.0 (1.35% with regard to the outer aqueous phase) and 10%, respectively. The gelling of the mixture of alginate solution plus multiple emulsion was induced by the addition of 26 mM CaSO4$2H2O with regard to the final gel mass. This Ca2C-salt and the addition of sodium polyphosphate were used in order to slow the gelling procedure. Mixing was conducted under vacuum (max. 200 mbar, Twister prow, Vacuum mixer, Renfert, Germany) to avoid the formation of air bubbles. The freshly prepared mixture (emulsion plus alginate solution in the presence of CaSO4) was loaded into a flat vessel with a cylindrical shape where the gel was allowed to set. Smaller cylindrical samples of the gel, 17 mm in diameter and 10 mm in height (w2 g), were removed directly from this vessel after 2 h of storage by using a cork borer. The storage temperature chosen was the same as the temperature at which the release study was performed after 2 h (23 and 7 8C). The rate of release of the encapsulated tryptophan was determined by the immersion of two of these gel samples in deionised water (ratio gel:water Z 1:5) for 24 h with gentle stirring (150 rpm). The release of tryptophan was measured and its release kinetics was determined at 23 and 7 8C. At defined intervals, samples of the surrounding medium were withdrawn and analysed for tryptophan concentration. The medium samples were then precipitated with an equal volume of 25% TCA followed by filtration and, if necessary, they were diluted. The tryptophan concentration was determined spectrophotometrically at 280 nm. 2.7. Statistics Duplicate analyses of all experiments were done. Statistical analysis was performed by means of STATGRAPHICSw 5 Plus version 5.1 using Duncan analysis (UMEX GmbH Dresden, Germany). Differences were considered significant at P!0.05.

3. Results 3.1. Characterization of multiple emulsion 3.1.1. Particle size of W/O and the multiple emulsion In Table 2 the average droplet diameters of the primary water-in-oil (W1/O) and the final water-in-oil-in-water emulsions (O-droplets) with respect to the type of lipid phase and the preparation conditions are listed. Fig. 1 shows the particle size distributions of the primary and final emulsions depending on the system composition. The smallest W1 particles of primary W/O emulsions were obtained using Chocosine with 8% PGPR as lipid phase (d43Z0.78 mm) and differed significantly from the droplet sizes of all other emulsions. The small size of these particles can be attributed to the lower surface tension between the inner water phase and the oil phase (data not shown). In contrast, the largest droplets were found in emulsions with Witocan and 2% PGPR (Table 2 and Fig. 1), whose phases showed the highest surface tension among the different emulsions. Remarkable small multiple emulsion droplets were obtained using Hardstock as either the sole lipid phase or a blend with MCT-oil (Table 2). In the latter case, the d32 value was in particular very small. Emulsions with the MCT-oil and 8% PGPR yielded the largest droplets. They were larger than the other formulations and this difference was statistically significant (except compared to the d43 of emulsions with Chocosine or Crokcool, both with 2% PGPR, Table 2 and Fig. 1). 3.1.2. Release of tryptophan during emulsion preparation and storage Immediately after homogenization the release of tryptophan into the outer phase of the multiple emulsions reached values between 5 and 15%, depending on the composition of the lipid phase (Table 3). Homogenization by means of a modified orifice valve system at low pressure allowed for the preparation of multiple emulsions with a high encapsulation efficiency (Scherze and Muschiolik, submitted).

Table 2 Average droplet diameter of W/O and O of W/O/W depending on the type of the lipid phase and the amount of lipophilic emulsifier Emulsion system composition of the lipid phase MCT-oil C8% PGPR MCT-oil C2% PGPR ChocosineC8% PGPR ChocosineC2% PGPR WitocanC2% PGPR Witocan (25/75)C2% PGPR CrokcoolC2% PGPR Crokcool (25/75)C2% PGPR HardstockC2% PGPR Hardstock (25/75)C2% PGPR

W1 droplets

O-droplets of W/O/W

d43 (mm)

d32 (mm)

b

b

1.12 1.13b 0.78a 1.18b 1.75e 1.41c,d 1.15b 1.44c,d,e 1.40b,c 1.46d,e

0.95 1.09b 0.70a 1.05b,c 1.71f 1.30c,d,e 1.09b 1.35d,e 1.19b,c,d 1.37e,f

d43 (mm) d

12.08 7.67a 7.19a,b 10.36c,d 7.90a,b,c 8.25a 8.77a 9.99b,c,d 6.05a 6.56a

d32 (mm) 9.73e 6.23c,d 6.15c,d 7.75d 6.35c,d 6.13b,c 6.09b,c 6.34c,d 4.50b 2.89a

The ratio 25/75 in parentheses means the amount of fat in the fat/MCT-oil blend (25% of fat within the blend). (a,b,.) Values with the same letter within a column are not significantly different.

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Fig. 1. Particle size distribution of W/O (A and C) and (W/O)/W (B and D) of the multiple emulsions prepared for embedding in a gel. (A and B) Effect of the type of lipid used for emulsion preparation. (C and D) Effect of the fat content in the lipid phase. The lipid phase of the double emulsion contains 2% PGPR. Legend: (%) Choc, (,) Crok, (*) Hard, (6) MCT, (:) Wit (100% fat in the oil phase of the emulsion), (B) Crok/MCT, ($) Hard/MCT, (&) Wit/MCT (25% fat in the oil phase of the emulsion).

Immediately after emulsification the lowest release occurred in emulsions with MCT-oil or Chocosine, when they were prepared with 8% PGPR (Table 3). Under the conditions of this investigation the reduction of the PGPR concentration (from 8 to 2%) in the emulsions prepared with MCT-oil or Chocosine led to a slight but not significant increase in the release of tryptophan during emulsion preparation (Table 3). In emulsions with 8% PGPR no additional leakage of tryptophan occurred during the first 24 h. In the case of emulsions with Chocosine and 2% PGPR, however, a significant increase in diffusion-controlled release occurred during storage. By comparing the extent of release of all emulsion formulations after 24 h of storage the lowest release was found in systems with 8% PGPR. When the PGPR concentration was decreased, the release was significantly higher (Table 3). Because of the only slight effect of the PGPR concentration on the encapsulation efficacy of tryptophan during emulsion preparation, the other fats and blends were all prepared with 2% PGPR. A lower PGPR concentration is, in fact, advantageous because the concentration will not exceed the maximum allowed level of PGPR. Comparing the lipid phases used, emulsions with Hardstock showed the highest release of tryptophan during emulsion preparation. A relative high release was also observed in the emulsions which consisted of blends of a fat with MCT-oil as the lipid phase (2% PGPR).

Storage had an adverse effect particularly on multiple emulsions with Crokcool (2% PGPR), in which the release increased up to 24% (in contrast to 10% immediately after emulsion preparation; Table 3). A comparatively high release also occurred from emulsions containing blends of MCT-oil and fat (Witocan, Hardstock, Crokcool), and from emulsions containing the fats Chocosine and Hardstock and lacking MCT-oil. Table 3 Release of the multiple emulsions with respect to the composition of the lipid phase of the emulsion Emulsion system composition of the lipid phase

Release (%) after preparation

Release (%) after storage for 24 h

MCT-oil C8% PGPR MCT-oil C2% PGPR Chocosine C8% PGPR ChocosineC2% PGPR WitocanC2% PGPR Witocan (25/75)C2% PGPR CrokcoolC2% PGPR Crokcool (25/75)C2% PGPR HardstockC2% PGPR Hardstock (25/75)C2% PGPR

5.5e 7.8d,e 5.4e 7.5d,e A 7.7d,e 13.6a,b 9.7c,d A 14.8a 11.3b,c 11.9a,b,c

5.4f 11.4e 6.6f 19.6b B 13.4d,e 18.0b,c 23.9a B 15.9c,d 15.9c,d 16.1c

The ratio 25/75 in parentheses means the amount of fat in the fat/MCT-oil blend (25% of fat within the blend). (a,b,.) Values with the same letter within a column are not significantly different. (A,B) Values with the same letter within a line are not significantly different.

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Fig. 2. Scanning electron micrographs of emulsion gels made with the MCT-oil. Oil volume fraction 25%, surfactant at the oil interface: WPI (0.625% within the outer aqueous gel phase). Magnification 400 and 2500 times, respectively.

3.2. Release properties of the emulsion alginate gels The release behaviour of the model substance L-tryptophan out of the emulsion gel matrices with variable composition was studied in pure water at two different storage temperatures, 7 and 23 8C. Different SEM images of an emulsion gel containing MCT-oil as the lipid phase are depicted in Fig. 2. The oil globules embedded within the alginate matrix are very well discernable. The addition of multiple emulsion to the alginate gel had no adverse effect on the gel formation process, which was confirmed by rheological measurements (data not shown; Weib, Scherze, Muschiolik, & Bindrich, 2004). 3.2.1. Effect of PGPR The PGPR concentrations chosen for these experiments were 2 and 8%, respectively. The lipid phases for preparing the multiple emulsions were MCT-oil and Chocosine. The release experiments were done at ambient temperature (23 8C). The results suggest that the PGPR concentration (2 or 8%) had no effect on the extent and rate of the marker release out of the emulsion gels, containing MCT-oil (Table 4, Fig. 3B). In the case of Chocosine, the release was lower if the lipid phase contained 8% PGPR (significant after 30 and 60 min). 3.2.2. Effect of lipid phase composition of the multiple emulsion and temperature on tryptophan release In this study, five different lipids were used to prepare the multiple emulsions with regard to their ability to function as a hydrophobic barrier between the two aqueous compartments for the release of the LMWS tryptophan. Therefore, various vegetable fats and a MCT-oil were tested, and the results were compared to the release from gels without the addition of a multiple emulsion. The fats possess different properties, in particular with respect to the fatty acid composition (Table 1) as well as melting point and melting behaviour. Due to the predicted high impact of the solid fat content of the lipid on the encapsulation efficacy (Roberts, Pollien, & Watzke, 2003), the release experiments were carried out at two different temperatures (7 and 23 8C).

Our experiments confirmed that temperature affects the diffusion kinetics of tryptophan release (Fig. 4A, Table 5). At almost all of the intervals of sample collection (storage for 30 min to 24 h), a reduction in release rate was observed when the environmental temperature was lowered from 23 to 7 8C. The most pronounced effect on release due to temperature reduction was observed in gels containing Chocosine. Here a very strong decrease in marker diffusion out of the gels was noted during the investigated storage period. A reduction in release rate at low temperature was also observed with Crokcool and the MCT-oil (Fig. 4A, Table 5). In contrast, in emulsion gels containing either Hardstock or Witocan, storage temperature had little effect on the extent of tryptophan diffusion. During an immersion time of 2 h in water the gel samples prepared with Chocosine exhibited the slowest release of tryptophan (at 7 8C, Table 5). A comparably low but slightly higher marker release resulted with the use of Witocan or Crokcool at 7 8C. They did not differ statistically significant from the release observed for gels with Chocosine (Table 5). The highest diffusion was obtained when the emulsion gels were prepared with Hardstock or MCT-oil at a temperature of 23 8C (Table 5, Fig. 4A). These effects are confirmed by comparing the diffusion coefficients calculated from the release during the first 120 min of the experiment. Compared to that, as previously predicted, the diffusion in Table 4 Effect of PGPR concentration on the release of tryptophan (%) from emulsion gels into the surrounding pure water Sample characteristics

Release of tryptophan (%) after time interval

Lipid phase composition

wRel (8C)

30 min

60 min

120 min

24 h

Without emulsion MCTC8% MCTC2% ChocC8% ChocC2%

23

32.4c

44.1d

60.5c

85.0a,b

23 23 23 23

29.0b 29.4b,c 23.3a 27.1b

39.0c 39.8c 29.6a 35.7b

51.7b 53.4b 41.3a 47.9a,b

89.3b 87.6a,b 76.0a 83.1a,b

wRel, Release temperature. Samples with 10% maltodextrin in the outer alginate gel phase. The value behind the lipid means the PGPR concentration. (a,b,.) Values with the same letter within a column are not significantly different.

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Fig. 3. Release rate of tryptophan from emulsion gels as a function of the square root of time (t0.5). (A) Effect of the fat type and content in the lipid phase. Lipid phase with 2% PGPR, environmental temperature 7 8C. (B) Effect of the PGPR concentration within the lipid phase; temperature 23 8C. Legend: (%) Choc, (,) Crok, (*) Hard, (6) MCT, (:) Wit, (B) Crok/MCT, ($) Hard/MCT, (&) Wit/MCT (all with 2% PGPR), (C) Choc 8% PGPR, (C) MCT 8% PGPR.

gels without the addition of multiple emulsion (that is, without a hydrophobic diffusion barrier) was higher than in the other samples, but after 24 h the amount released from gels with a multiple emulsion was the same as the amount released from gels without a multiple emulsion (Table 5). 3.2.3. Effect of lipid blends (W/O/W in emulsion gel) In order to investigate the influence of the fat content of the hydrophobic phase of the double emulsion, emulsions were formulated with varying fat contents. The lipid phase consisted of either 100 or only 25% fat (Witocan, Crokcool, and Hardstock) with both containing 2% of the lipophilic emulsifier PGPR. The release temperature was 7 8C. A significant effect on the extent of marker diffusion was observed by varying the fat content. During the time period of the investigation the lower fat content (25%) emulsion caused an increase in release as compared to the system with a fat content of 100% (Figs. 3A and 4B, Table 6), independent of the kind of fat used to prepare the multiple emulsion. Fig. 4 summarizes the cumulative release of tryptophan out of emulsion gels varying in composition recorded over a storage period of 24 h in pure water. The values represent

the amount of tryptophan released from the sample gels compared to the amount released out of control alginate gels prepared from a simple polysaccharide solution with maltodextrin lacking the double emulsion (temperature 23 8C; release value presumed to be 100%). A higher release was observed in gels which contained a double emulsion with a blend of Hardstock and MCT-oil (cumulative release 105%, Fig. 4B). Even the system containing pure MCT-oil (including 2% PGPR) showed a comparably lower relative release of the marker (94%, Fig. 4A).

4. Discussion The finding that the release in the case of the double emulsion is very low and in the case of the emulsion gel is higher can be attributed to distinct differences in the treatment of the two systems. The greatest impact on the release of the marker from the emulsion gels was the storage in water, where the ratio between water and gel sample was 5:1. This means that a new osmotic balance had to be redressed between the water phases (W1 and the outer water phase including the aqueous alginate gel). It is likely that

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Fig. 4. Relative release of tryptophan (%) out of the emulsion gels compared to gels prepared without multiple emulsion (Z100% during a period of 24 h). (A) Effect of the lipid phase at an environmental temperature of 23 8C. (B) Effect of the type of lipid and the fat content in the lipid phase at a temperature of 7 8C. All systems contained 2% of PGPR in the lipid phase. Table 5 Effect of the lipid phase composition and the temperature on the release of tryptophan (%) from emulsion gels into the surrounding pure water Sample characteristics

Release of tryptophan (%) after time interval

Lipid phase composition

wRel (8C)

30 min

60 min

120 min

24 h

Without emulsion MCT MCT Choc Choc Wit Wit Crok Crok Hard Hard

23

32.4f

44.1e

60.5f

85.0e,f

23 7 23 7 23 7 23 7 23 7

29.4d,e,f 25.7b,c 27.1c,d 21.9a 25.8b,c 23.1a,b 25.7b,c 24.9a,b,c 31.3e,f 28.4c,d,e

39.8d,e 33.8a,b,c,d 35.7b,c,d 27.5a 32.8a,b,c 34.4b,c,d 32.4a,b 31.3a,b 36.8b,c,d 39.3c,d,e

53.4d,e 44.1b,c 47.9c,d 36.0a 38.7a,b 39.5a,b 48.7c,d 43.5b,c 54.9e,f 49.1c,d,e

87.6f 78.3d,e,f 83.1d,e,f 65.1a,b,c 59.9a 60.9a,b 86.1f 72.8b,c,d 72.2b,c,d 73.6c,d,e

wRelZRelease temperature. Samples with 10% maltodextrin in the outer alginate gel phase. All emulsions contained 2% PGPR in the lipid phase. (a,b,.) Values with the same letter within a column are not significantly different.

Table 6 Effect of the lipid blend on the release of tryptophan (%) from emulsion gels into the surrounding pure water Sample characteristics

Release of tryptophan (%) after time interval

Lipid phase composition

wRel (8C)

30 min

60 min

120 min

24 h

Without emulsion Wit Wit (25) Crok Crok (25) Hard Hard (25)

23

32.4c,d

44.1c

60.5e

85.0c

7 7 7 7 7 7

23.1a 26.4a,b 24.9a,b 28.4b,c 28.4b,c 36.2d

34.4a,b 36.2b 31.3a 38.1b 39.3b 45.3c

39.5a 46.1a,b,c 43.5a,b 52.8c,d 49.1b,c 59.2d,e

60.9a 78.2b 72.8b 85.3c 73.6b 93.3d

wRelZRelease temperature. Samples with 10% maltodextrin in the outer alginate gel phase. Value in parentheses means the fat content in the lipid phase in blends with MCT-oil, all emulsions contained 2% PGPR in the lipid phase. (a,b,.) Values with the same letter within a column are not significantly different.

J. Weiss et al. / Food Hydrocolloids 19 (2005) 605–615

water from the release medium diffused into the W1 phase and caused a considerable swelling of the W1 droplets ¨ zer, Baloglu, Ertan, followed by their breakdown (O Muguet, & Yazan, 2000; Jager-Lezer et al., 1997). This would favour a higher diffusion of tryptophan. Such storage in water was not applied to the multiple emulsions without the alginate matrix which were investigated in the present study. Hence, the release was limited in the multiple emulsions (Table 3) due to their composition which promotes a high stability. With such a treatment, instability of the very stable multiple emulsion was caused when the emulsion gels (containing the multiple oil globules) were stored in water. Storage in water was necessary to observe any differences in diffusion of tryptophan caused by the lipid phases used in this study. The limited release of the low molecular model substance tryptophan from double emulsion during preparation and partially during storage (Table 3) was mainly due to the presence of sodium chloride in W1 ([NaCl]Z0.1 mol lK1). Hino et al. (2001) reported that an increase in encapsulation of tryptophan in W/O/W emulsions occurred as the concentration of sodium chloride added to the inner water phase (0–1.0 mol lK1 NaCl) was increased. In our experiments, immediately after the preparation of a multiple emulsion, a rapid thickening of the emulsions was observed due to the water flux from the W2 phase to the W1 phase which caused swelling of the droplets. In general, the encapsulation efficiency of the investigated systems (multiple emulsion and emulsion gel) was limited by the risk of swelling of the W1 droplets as well as oil globules leading to rupture followed by the marker release. The leakage from emulsion gel systems was not only controlled by this event but also by the diffusion through the gel. A high concentration of lipophilic surfactant is known to increase the swelling capacity of the oil globules ¨ zer, Baloglu et al. (2000) (Jager-Lezer et al., 1997). O postulated that the more the oil globules swell due to an elevated concentration of lipophilic polymeric surfactant, the less hydrophilic substance will be released. In our investigations, during emulsification the PGPR content had no essential effect on the release. But as to be seen from the release after 24 h of storage the lower PGPR content increased the tryptophan leakage significantly (MCT-oil, Chocosine, Table 3). These results are in agreement with the assumption that the potential of the swelling capacity on encapsulation becomes considerably more important during storage periods of multiple emulsion systems. A significant reduction in encapsulation efficiency (during emulsion preparation and after storage) was obtained by the addition of more liquid oil phase (75 vs. 0%). However, the low release in emulsions containing 100% liquid oil (including PGPR) compared to the release in systems using the fat blends seems to indicate a disadvantageous interaction between the fatty acid chains

613

of the fats and the MCT-oil in all the blends. This could have favoured instability of the double emulsion, since, under the low cooling rate of the present study, the crystals in the blend could have grown more than the crystals in the more compact and viscous lipid phase containing 100% fat. Hence, the larger crystals in the blend could have disrupted the thin oil layer between the water phases. In contrast to the results obtained by Garti et al. (1999) the crystals within the emulsion gels in our experiments were not of submicron size and could, therefore, have destabilized the double emulsion. The relatively rapid release of tryptophan out of the emulsion gels into the suspending medium can be, on the other hand, explained by the fact that the inner water droplets and the oil globules possess a large surface area. Such large interfaces favour a rapid exchange of uncharged LMWS (Collings, 1971; Dickinson et al., 1994; Nakhare & Vyas, 1997; Okochi & Nakano, 2000; Omotosho, Whateley, Law, & Florence, 1986; Owusu, Zhu, & Dickinson, 1992), especially when the instability of the multiple emulsion is promoted by the storage in water. This applies to all the investigated formulations because each kind of emulsion possessed a large interface between the phases. However, in our investigations, there was no correlation between the W1 particle size and the release rate of tryptophan. The differences in release from emulsion gels and their corresponding W1 particles, which also differed in size to a certain degree, thus, must be affected by other factors which are related to the composition of the lipid. The composition can have a pronounced impact on the encapsulation efficacy by influencing the stability of the system which plays a crucial role in release of the marker. This has to be further investigated. Besides this, the type of lipid affects the partition coefficient of the marker between the phases (Nakhare & Vyas, 1995) as well as the solubility of the marker within the lipid. Omotosho et al. (1986) showed a correlation between the release and the type and polarity of the oil phase of a multiple emulsion. Therefore, the differences in release rate among the samples could be ascribed to the composition of the lipid phase, taking into consideration the effect of the temperature on their physical parameters (Table 1). In fact, the composition of the outer alginate phase was always the same, only the lipid phase varied in fatty acid composition and physical state. There are various factors that affect the instability of the double emulsion and thus the release of the marker from the encapsulation systems investigated here. These include the tryptophan leakage from W1 to W2 phase during double emulsion preparation, leakage during the gel preparation process, and leakage during storage of the gel samples in water during the release experiment. These factors are wellknown and suggest that a relatively high amount of the originally encapsulated tryptophan was present in the outer aqueous phase. In the case of systems with fat, tryptophan

614

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could have diffused into the outer aqueous phase prior to crystallization and prior reaching the temperature dependent solid fat content. This could, partially, explain the apparently high release which occurred from emulsion gels containing a high amount of fat. The cooling-conditions applied in the present study, however, had a smaller effect on the release than expected. In general, the embedding of multiple emulsions in alginate gels resulted in a more prolonged release of the LMWS compared to gels without emulsion (see Tables 4–6). The lower release observed in emulsion gels containing Chocosine with 8% PGPR (compared to Chocosine with 2% PGPR) can be attributed to the higher swelling capacity of the inner water droplets. The high ratio of water and gel sample (5:1) was expected to result in the breakdown of the W1 droplets followed by the release of the marker. The higher resistance against swelling and breakdown in systems with 8% PGPR could therefore partially prolong the release. The fact that the release decreased with decreasing environmental temperature can be explained by assuming that the solid fat content (SFC) increases as the temperature decreases. With increasing SFC the transport of water and the marker across the thin lipid layer is limited due to the formation of a crystalline lipid network. A higher SFC in an encapsulation system was found to lower the diffusion of entrapped components (Roberts et al., 2003). Moreover, the higher the SFC the more crystals are present at both interfaces and in the oil phase, which leads to an obstruction of the diffusion paths of the entrapped molecule. The SFC varies not only with temperature (for a particular fat), but also varies between different fats. Thus, Witocan (highest melting point), Crokcool and Chocosine have a higher SFC than Hardstock (p. e. at 10 or 25 8C, see Table 1). Despite the fact that Chocosine has a lower SFC at 20 8C than Crokcool, the release was slightly lower in the first case. Accordingly there existed no clear correlation between SFC and encapsulation. Roberts et al. (2003) investigated the effect of different lipid phases for preparing an O/W emulsion on the encapsulation of aroma compounds. They found differences in release of these compounds depending on the lipid type and ascribed their findings to the ability of the lipid to adsorb such substances to varying extent. The release decreased with increasing solubility of the aroma compound in the oil phase. Therefore, in our experiments, an effective adsorption of the highly water-soluble tryptophan to the lipids can not be expected. As in the W/O/W emulsion, the lower fat (25 vs. 100%) was associated with a higher release from the gels. In contrast, the relatively marked reduction in release rate by temperature reduction observed in gels prepared with MCT-oil can be explained by the increase in the viscosity of the alginate gel network at the lower temperature. This could have caused the marked decrease

in release. Results obtained from gels without emulsion confirms this assumption since lowering the temperature also led to a decrease in release rate (data not shown). As expected, diffusion, therefore, is also a function of temperature and a reduction in temperature causes a diminished diffusion rate. In spite of that, the actual observed drop in release rate did not match the expected one. Hence, we have to conclude that the leakage of tryptophan was even larger at low temperatures than at room temperature. This can, at least partly, attributed to the formation of larger crystals in the case of fat containing emulsion gels at reduced temperature, which were able to disrupt the oil membrane.

5. Conclusions In emulsion gels containing a W/O/W prepared with different fats, which were cooled for crystallization, only slight differences could be observed depending on the type and properties of the lipid phase. With the tested systems it was not possible to considerably alter the transportation of the marker to the storage solution. However, it is difficult to control the crystallization process of the lipid phase in freshly prepared double emulsions during cooling. When applying the cooling conditions to the emulsion gels, which were investigated in the present study, crystals were formed in the lipid phase of the double emulsion, but in an uncontrolled manner. These cooling conditions were unfavorable to the release properties of the emulsions. Under the experimental conditions the lowest release was achieved by using the fat with the highest melting point (Witocan). Almost independent of the composition of the system, the temperature reduction had a relatively high impact on marker release, since the release decreased with temperature (from 23 to 7 8C). Future experiments will examine the release profile of emulsion gels as encapsulation systems with a liquid oil phase enriched with submicron crystals. The crystallization process in this case will occur within the oil phase prior to emulsification (Garti et al., 1999; Garti, Binyamin, & Aserin, 1998).

Acknowledgements This research project was supported by the FEI (Forschungskreis der Erna¨hrungsindustrie e.V., Bonn), the AiF and the Ministry of Economics and Labour, Project No: AiF-FV-13064 BR and AiF-FV-13393 BR II. Dr. Ute Bindrich (DIL e.V., Quakenbru¨ck, Germany) is thanked for the realization of SEM analysis.

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