Sodium caseinate–maltodextrin conjugate stabilized double emulsions: Encapsulation and stability

Food Research International 43 (2010) 224–231

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Sodium caseinate–maltodextrin conjugate stabilized double emulsions: Encapsulation and stability Jonathan O’Regan, Daniel M. Mulvihill * Department of Food and Nutritional Sciences, National University of Ireland Cork, University College Cork, Cork, Ireland

a r t i c l e

i n f o

Article history: Received 3 July 2009 Accepted 22 September 2009

Keywords: Protein–polysaccharide conjugates Sodium caseinate Maltodextrin Double emulsions Encapsulation properties

a b s t r a c t The potential food applications of water-in-oil-in-water (W1/O/W2) double emulsions are great, including the encapsulation of flavours or active ingredients. However, the stability of these emulsions restricts their applications in food systems. Sodium caseinate (NaCN)–maltodextrin (Md40 or Md100) conjugates were investigated for their potential to improve the stability of W1/O/W2 double emulsions compared to NaCN. NaCN–Md40 and NaCN–Md100 conjugates were prepared by a Maillard-type reaction by dry heat treatment of mixtures of NaCN–Md40 or NaCN–Md100 at 60 °C and 79% relative humidity for 4 days. Water-in-oil-in-water (W1/O/W2) double emulsions with NaCN, NaCN–Md40 or NaCN–Md100 as outer aqueous phase containing emulsifier were prepared using a two-step emulsification process. General emulsion stability was characterised by determining the droplet size distribution, viscosity characteristics and by confocal microscopy of the W1/O/W2 double emulsions on formation and after their storage under accelerated shelf life testing conditions at 45 °C for up to 7 days. Inner phase encapsulation and stability were characterised by monitoring the level of entrapped Vitamin B12 in the inner aqueous phase on formation of the double emulsions and after storage at 45 °C for up to 7 days. Conjugate stabilized emulsions were more generally stable than NaCN stabilized emulsions. In comparison to NaCN stabilized emulsions, conjugate stabilized emulsions showed improved Vitamin B12 encapsulation efficiency in the inner aqueous phase on emulsion formation and improved encapsulation stability following storage of the emulsions. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Double emulsions are compartmentalised liquid dispersions in which the droplets of the dispersed phase contain smaller dispersed droplets that are similar to the continuous phase. The potential applications of double emulsions in the food industry are great, including the encapsulation of flavours or active ingredients, the production of low calorie and reduced fat products, the masking of flavours, the prevention of oxidation and the improvement of sensory characteristics (Muschiolik, 2007; Van der Graaf, Schroën, & Boom, 2005). There are two main types of double emulsions, (1) water-in-oil-in-water (W1/O/W2) emulsions in which an w/o emulsion is dispersed in a secondary aqueous phase and (2) oil-in-water-in-oil (O1/W/O2) emulsions in which an o/w emulsion is dispersed in a secondary oil phase. Water-in-oil-in-water (W1/O/ W2) double emulsions are the most commonly studied double emulsions because most food emulsions have an aqueous continuous phase and there is a greater availability/selection of food grade hydrophilic emulsifiers that can be used to stabilize the dispersed

* Corresponding author. Tel.: +353 21 4902650; fax: +353 21 4270001. E-mail address: [email protected] (D.M. Mulvihill). 0963-9969/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2009.09.031

oil phase (Pays, Giermanska-Kahn, Pouligny, Bibette, & LealCalderon, 2002). Water-in-oil-in-water (W1/O/W2) double emulsions contain three distinct phases; an inner aqueous phase (W1), which is encapsulated in an oil phase (O), which is enclosed within a second aqueous phase (W2). The major problems concerning W1/O/W2 double emulsions is that they tend to be difficult to prepare, particularly on an industrial scale, but the primary concern is their stability on manufacture, during storage and on exposure to environmental stresses such as mechanical forces, thermal processing, freezing or dehydration. These processes can lead to leakage of the inner aqueous phase (W1) and/or destabilization of the emulsion. There are four proposed mechanisms for the instability of W1/O/W2 double emulsions; these are: (1) coalescence of the inner aqueous droplets; (2) coalescence of the oil droplets; (3) rupture of the oil film separating the inner and outer aqueous phases and (4) migration/diffusion of water and water soluble materials from the inner and outer aqueous phases through the oil layer via reverse micellar transport (Appelqvist, Golding, Vreeker, & Zuidam, 2007, chap. 3; Florence & Whithill, 1981; Van der Graaf et al., 2005). Various different strategies have been employed to improve the stability of W1/O/W2 double emulsions including: increasing the viscosity of the outer aqueous phase (Özer, Baloglu, Ertan, Mugent, & Yazan, 2000); the addition of various

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emulsifier combinations to the aqueous phases and/or oil phase (Owusu Apenten & Zhu, 1996; Shima, Tanaka, Kimura, Adachi, & Matsuno, 2004; Su, Flanagan, Hemar, & Singh, 2006); the addition of oil-insoluble electrolytes to the inner aqueous phase (W1) to balance the Laplace and osmotic pressures between the inner aqueous phase and the outer aqueous phase (Garti, 1997; Kanouni, Rosano, & Haouli, 2002; Rosano, Gandolfo, & Hidrot, 1998); the solidification of the oil phase; the modification of the solubility and polarity of the oil phase to make it less water soluble (Tedajo, Seiller, Prognon, & Grossiord, 2001) and the incorporation of stabilizers (Benna-Zayani, Kbir-Ariguib, Trabelsi-Ayadi, & Grossiord, 2008) and biopolymers in the outer aqueous phase (W2) (Allouche, Tyrode, Sadtler, Choplin, & Salager, 2003; Benichou, Aserin, & Garti, 2004; Benichou, Aserin, & Garti, 2007). However, many of these strategies for improving the stability of W1/O/W2 double emulsions are not suitable for use in food applications because they are not easily scaled up, they are not cost effective or they may require the use of non-food grade ingredients making these double emulsions unsuitable for human consumption. Milk proteins such as sodium caseinate, whey protein isolate and bovine serum albumin have been added to the outer aqueous phase (W2) of double emulsions as secondary emulsifiers of the (water-containing) oil droplets (W1/O) (Garti, Aserin, & Cohen, 1994; Scherze & Fechner, 2007). However, many of these W1/O/W2 double emulsions tended to coalesce relatively quickly on storage, particularly at room temperature. The addition of low molecular weight emulsifiers in addition to these proteinaceous emulsifiers has been shown to increase stability of W1/O/ W2 double emulsions; however, high concentrations of these emulsifiers are required to stabilize these double emulsions and this may be problematic from a practical/industrial perspective as the concentration and type of low molecular weight emulsifiers permitted for use in food products are strictly regulated; low molecular weight emulsifiers also increase the final product cost. Milk protein ingredients in general, and caseinates especially, are very good fat emulsifiers and are widely used in emulsifying applications in foods (Dalgleish, 1997). While casein/caseinate ingredients are still relatively inexpensive, the rising cost of and functional demands on milk protein ingredients have created a need for speciality milk protein ingredients which possess the requisite physico-chemical and functional properties for specific food applications. One modification which is gaining acceptance as a potentially valuable modification to produce speciality ingredients is protein–polysaccharide conjugation via a Maillard-type reaction (Kato, 2002). Conjugates produced by this method have been shown to possess improved functional properties, including increased solubility, particularly around the isoelectric pH of the protein (Chevalier, Chobert, Popineau, Nicolas, & Haertlé, 2001; Jiménez-Castaño, López-Fandiño, Olano, & Villamiel, 2005) and enhanced emulsifying properties compared to the protein itself (Akhtar & Dickinson, 2003; O’Regan & Mulvihill, 2009a,b; Shepherd, Robertson, & Ofman 2000). Fechner, Knoth, Scherze, and Muschiolik (2007) prepared sodium caseinate–dextran (1:3, w/w) conjugates by the dry heating method (incubating the caseinate– dextran mixture at 60 °C, 79% RH for 8 h) and characterised the conjugates by SDS gel electrophoresis and determination of their solubility and interfacial properties. These sodium caseinate–dextran conjugates were added in place of caseinate to the outer aqueous phase as secondary emulsifiers in the preparation of neutral pH W1/O/W2 double emulsions; the conjugate stabilized double emulsions had smaller (water-containing) oil droplets with a narrower droplet size distribution in comparison to caseinate stabilized double emulsions; however, conjugation of the protein only slightly influenced the encapsulation of the inner aqueous phase marker, Vitamin B12.

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The objective of the research reported here was to investigate the potential of two types of sodium caseinate–maltodextrin conjugate to improve the general stability, the encapsulation efficiency and the encapsulation stability of W1/O/W2 double emulsions. Extensively conjugated caseinates that were well characterised (O’Regan & Mulvihill, 2009a) were used in this study, while more mildly conjugated caseinates that were less extensively characterised were used in the studies of Fechner et al. (2007). 2. Materials and methods 2.1. Materials Sodium caseinate (NaCN) containing 90.4% protein was obtained from Kerry Ingredients (Listowel, Co. Kerry, Ireland). The maltodextrins, Maltrin040 (Md40) and Maltrin100 (Md100) with Dextrose Equivalent (DE) of 4–7 and 9–12, respectively, were obtained from Grain Processing Corporation (Muscatine, IA, USA). The Mds were pre-dialysed against Milli-Q water (20 volumes, with 2 water changes) containing 0.01%, w/w, sodium azide at 4 °C for 48 h using 12 kDa dialysis membrane. The post-dialysis DE of the Mds was not determined. Sodium caseinate (NaCN)–maltodextrin (Md40 or Md100) conjugates were prepared by the dry heating method (incubating the dry mixture at 60 °C, 79% relative humidity for 96 h) as described previously (O’Regan & Mulvihill, 2009a). Gelatin (Type A: Bloom 75–100) was purchased from Sigma–Aldrich (Dublin, Ireland). Medium chain Triglyceride oil (MCT-oil, MIGLYOLÒ 810) was purchased from SASOL GmbH (Hamburg, Germany). The oil soluble emulsifier, polyglycerol polyricinoleate (GrindstedÒ PGPR 90 Kosher) was provided by Danisco A/S (Copenhagen, Denmark). Vitamin B12 was purchased from Sigma–Aldrich (Dublin, Ireland). Nile Blue was purchased from Sigma Chemicals (St. Louis, MO, USA). All other chemicals were analytical grade and were commercially available from Sigma–Aldrich (Dublin, Ireland). 2.2. Preparation of water-in-oil-in-water (W1/O/W2) emulsions Water-in-oil-in-water (W1/O/W2) emulsions were prepared using the modified two-step emulsification method described by Fechner et al. (2007). The inner aqueous phase (W1) was prepared by hydrating gelatine (5%, w/w, Bloom 75–100), NaCl (0.6%, w/w) and a water soluble/oil insoluble marker (Vitamin B12, 0.03%, w/ w, 100% water soluble, 0% oil soluble) in a sodium phosphate buffer (0.1 M, pH 7), containing sodium azide (0.02%, w/w), at 60 °C for 5 min using moderate magnetic stirring. Gelatine was added to solidify the inner aqueous phase (W1) of the double emulsion as it has previously been shown to increase the encapsulation efficiency and the oil droplet stability of W1/O/W2 double emulsions (Fechner et al., 2007; Muschiolik et al., 2006). The oil phase (O) was medium chain triglyceride (MCT) oil containing 2%, w/w, of the oil soluble emulsifier polyglycerol polyricinoleate (PGPR). The outer aqueous phase (W2) was prepared by hydrating NaCN or NaCN–Md40 or NaCN–Md100 conjugate [1%, w/w, protein, as determined by macro Kjeldahl (AOAC, 1995, chap. 33)] in a sodium phosphate buffer (0.1 M, pH 7) containing sodium azide (0.02%, w/ w) under moderate magnetic stirring conditions. A water-in-oil (W1/O) emulsion was prepared by mixing the inner aqueous phase (W1) (20%, w/w) with the oil phase (O) (80%, w/ w) and allowing the mixture to temper at 60 °C for 30 min; the mixture was then homogenised at 60 °C using an Ultra-Turrax operating at 20,000 rpm for 2 min. The W1/O emulsion (40%, w/ w) was gradually added to the outer aqueous phase (W2) (60%, w/w), mixed using an overhead stirrer at 1500 rpm for 2 min and homogenised (APV 1000 Homogeniser, APV AS, Albertslund,

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Denmark) at 60 °C for three passes using a first stage pressure of 15 MPa and a second stage pressure of 5 MPa to produce the final W1/O/W2 (8:32:60, w:w:w) double emulsion. 2.3. Determination of W1/O/W2 double emulsion stability

2.3.1. Emulsion droplet size General double emulsion stability, or the ability of the emulsions to resist changes in the size distribution and mean size of (water-containing) oil droplets (W1/O), was characterised by measuring the size distribution and the mean size of the (water-containing) oil droplets (W1/O) dispersed in the outer aqueous phase (W2) of each W1/O/W2 double emulsion, on emulsion formation and after storage of the emulsion in a glass container under accelerated shelf life testing conditions of 45 °C for 7 days (Lynch & Mulvihill, 1997). The droplet size distribution was measured by dynamic light scattering using a Malvern Particle Size Analyser (Mastersizer 2000S, Malvern Instruments Ltd., UK) equipped with a He–Ne laser (k = 623 nm). The optical parameters selected were, a dispersed phase refractive index of 1.446, a droplet absorbance of 0.001 and a continuous phase refractive index of 1.333. Droplet size measurements were carried out in duplicate on each emulsion and the results are reported as the typical droplet size distribution (size range measured from 0.05 to 878.67 lm), and the typical volume weighted mean droplet size, d43:

P 4 ni di d43 ¼ P 3 ni di

where ni is the number of droplets of a diameter di. 2.3.2. Confocal scanning laser microscopy Confocal scanning laser microscopy (CSLM) (Leica TCS SP5, Leica Microsystems, Germany) was used to estimate the size of the water droplets in the W1/O emulsion and also to study the general stability of W1/O/W2 double emulsions on storage at 45 °C for 7 days. CSLM was carried out by taking 1 ml of (i) the W1/O emulsion and mixing with 2 ml of MCT oil or (ii) the W1/O/W2 double emulsion and mixing with 2 ml of sodium phosphate buffer (0.1 M, pH 7). All samples were stained for both protein and fat by thoroughly mixing with 2 ll Nile Blue (0.1%, w/v); 10 ll of sample was then dropped on a plain glass slide (Superfrost, VWR International, Leuven), covered by a cover slip and examined with a 63 objective oil immersion, with a zoom factor of 5. The protein and fat phases were confocally illuminated simultaneously using an Argon laser (488 nm excitation) and a Helium–Neon laser (633 nm excitation), and the emissions were collected in the wavelength range of 510–590 nm and 650–759 nm, respectively. 2.3.3. Emulsion viscosity The viscosity of each W1/O/W2 double emulsion was determined using a CarriMed CSL-100 rheometer (TA Instruments, Surrey, UK) as follows: W1/O/W2 double emulsion (12 ml) was transferred to the double-concentric-cylinder measuring system and allowed to equilibrate to 20 ± 1 °C for 10 min prior to analysis. The sample was sheared by increasing the shear rate from 0 to 300 s1 over a 75 s period followed by holding the shear rate constant at 300 s1 for 75 s. The viscosity (mPa s) of each emulsion was recorded as a function of shear time; the mean constant shear viscosity was determined from the data obtained at 300 s1. 2.4. Determination of encapsulation efficiency and encapsulation stability of the W1/O/W2 double emulsions The encapsulation properties of W1/O/W2 double emulsions are often characterised in terms of encapsulation efficiency and encap-

sulation stability. Encapsulation efficiency can be defined as the percentage of the aqueous phase marker added to the inner aqueous phase (W1) which remains entrapped in the inner aqueous phase (W1) on manufacture of the W1/O/W2 double emulsion while encapsulation stability can be defined as the percentage of the aqueous phase marker added to the inner aqueous phase (W1) which remains entrapped in the inner aqueous phase (W1) following storage or on exposure of the W1/O/W2 double emulsion to environmental stresses. In order to accurately determine the encapsulation efficiency and encapsulation stability of the W1/O/ W2 double emulsions, the accuracy of the spectrophotometric measurement of the level of Vitamin B12 marker in the aqueous phase of the emulsion was determined by measuring the percentage of Vitamin B12 marker measurable in the outer aqueous phase (W2), while the ability to recover all of the Vitamin B12 marker added to the outer aqueous phase of an emulsion in the subnatant obtained following centrifugation of the emulsion was determined by measuring the recovery yield as described previously (O’Regan & Mulvihill, 2009c). The percentage of the Vitamin B12 marker measurable by spectrophotometry in the outer aqueous phase of the emulsion was found to be 100% while the recovery yield of Vitamin B12 marker in the centrifugal subnatant was found to be >99%. Therefore, the encapsulation efficiency and encapsulation stability of the NaCN, NaCN–Md40 and NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions were determined using the same methods/conditions as described previously (O’Regan & Mulvihill, 2009c). Briefly, an aliquot (5 g) of the W1/O/W2 double emulsion was diluted with an aliquot (5 g) of phosphate buffer (0.1 M, pH 7); the mixture was gently inverted and centrifuged at 30,000g at 4 °C for 30 min, after which the subnatant was carefully removed and filtered using a 0.2 lm Whatman filter paper. The concentration of Vitamin B12 marker in the filtered subnatant (recovered outer aqueous phase, W2) was determined by measuring its absorbance at 361 nm. The encapsulation efficiency (EE, %) was then calculated using Eq. (1). After manufacture, each NaCN, NaCN–Md40 conjugate and NaCN–Md100 conjugate stabilized W1/O/W2 double emulsion was stored in a glass container under accelerated shelf life testing conditions at 45 °C for up to 7 days after which time the concentration of the Vitamin B12 marker in the outer aqueous phase (W2) was again determined as described above. The encapsulation stability (ES, %) was then calculated using Eq. (2).

EE ð%Þ ¼ 100  ðCmOAP  100=RyÞ

ð1Þ

ES ð%Þ ¼ 100  ðCmOAPðtÞ  100=RyÞ

ð2Þ

CmOAP is the percentage of total Vitamin B12 marker added to the inner aqueous phase of the double emulsion that is present in the outer aqueous phase recovered by centrifugation following preparation of the W1/O/W2 double emulsion. CmOAP(t) is the percentage of total Vitamin B12 marker added to the inner aqueous phase of the double emulsion that is present in the outer aqueous phase recovered by centrifugation following storage of the W1/O/W2 double emulsions for 7 days at 45 °C. Ry (%) is the measured recovery yield for the marker. 2.5. Statistical analysis All experiments were carried out in triplicate and results are expressed as mean ± standard deviations. The encapsulation efficiency and encapsulation stability data were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s mean comparison test to establish the significance of differences among the mean values at p < 0.05. The statistical analyses were performed using Minitab Release version 13 (State College, PA, USA).

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3. Results and discussion 3.1. Conjugate characteristics The characteristics of the NaCN and of the NaCN–Md40 and NaCN–Md100 conjugates used have been described previously (O’Regan & Mulvihill, 2009a). In brief, the NaCN used had 90.4% protein while the conjugates had equal proportions by weight of NaCN and Md, and thus had a protein content of 45.2%. Conjugation resulted in a loss of 35.6% and 36.2% of the available amino groups in the protein for the NaCN–Md40 and NaCN–Md100 conjugates, respectively, and a loss of 17.8% and 25.7% of the available reducing groups in the pre-dialysed Md for the NaCN–Md40 and NaCN–Md100 conjugates, respectively. While a significant amount of the protein is conjugated to the Md, it must also be noted that a portion of Md remained unreacted in each conjugate. 3.2. General W1/O/W2 emulsion stability 3.2.1. Droplet size characteristics Immediately after preparation, the W1/O/W2 double emulsions stabilized by NaCN, NaCN–Md40 and NaCN–Md100 conjugates displayed similar bimodal droplet size distributions (Fig. 1a) with a population of droplets of diameters <1 lm and a population of droplets of diameters >1 lm and <20 lm. On formation, the NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions had the smallest mean droplet size of 1.9 lm (a larger population of droplets of diameter <1 lm) in comparison to the NaCN and NaCN–Md40 conjugate stabilized W1/O/W2 emulsions which had mean droplet size of 2.5 lm and 3.2 lm, respectively (Fig. 1a). Su et al. (2006) observed similar droplet size distributions for NaCN stabilized W1/O/W2 double emulsions. On storage of the emulsions at 45 °C for 7 days, the NaCN stabilized double emulsion was the least stable (Fig. 1b); there was a reduction in the population of droplets of diameters <1 lm and an increase in the population of droplets of diameters between 1 lm and 100 lm, with the mean droplet size typically increasing from 2.5 to 11.0 lm. There was also a shift in the droplet size distribution and mean droplet size of the NaCN–Md40 conjugate stabilized emulsions; again there was a reduction in the population of droplets of diameters <1 lm and an increase in the population of droplets of diameters between 1 lm and 100 lm with the mean droplet size (d43) typically increasing from 3.2 to 9.0 lm. In comparison to the NaCN and NaCN– Md40 conjugate stabilized double emulsion, the NaCN–Md100 conjugate stabilized double emulsion exhibited minimal change in droplet size distribution and only a slight increase in the mean droplet size from 1.9 to 2.6 lm. These results show that both NaCN–Md conjugates stabilized W1/O/W2 double emulsions had improved stability, in particular the NaCN–Md100 conjugate stabilized double emulsion, when compared to the NaCN stabilized double emulsion. This improved general emulsion stability may be attributed to the conjugated protein molecule forming a more bulky polymeric layer than the non-conjugated protein on the droplet surface, with the Md portion protruding outwards into the continuous phase providing better steric stabilization, thus preventing droplet aggregation and coalescence (Akhtar & Dickinson, 2003; Fechner et al., 2007). In addition, the conjugate stabilized emulsions also had an appreciable level of non-conjugated Md in the continuous phase contributing to enhanced continuous phase viscosity resulting in reduced Brownian motion of the droplets (O’Regan & Mulvihill, 2009a,b), thus reducing droplet collision frequency and improving storage stability (McClements, 2004).

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3.2.2. Confocal scanning laser microscopy images of emulsions CSLM images of the primary W1/O emulsion and the final W1/O/ W2 double emulsions stabilized with NaCN, NaCN–Md40 and NaCN–Md100 conjugate are shown in Fig. 2. On formation of the W1/O emulsion (Fig. 2i, a) the inner aqueous phase droplets (W1) were uniformly distributed in the oil phase with the majority of the droplets observed to be less than 1 lm in diameter (estimated by reference to the scale bar). On storage of the primary W1/O emulsion for 7 days at 45 °C, there was some increase in the size of the inner aqueous phase droplets (W1) (Fig. 2i, b) but the droplets still remained as individual droplets uniformly distributed in the oil phase. These results suggest that the primary W1/O emulsion was relatively stable on storage for 7 days at 45 °C. CSLM images confirmed that W1/O/W2 double emulsions stabilized with NaCN (Fig. 2ii), NaCN–Md40 conjugate (Fig. 2iii) and NaCN–Md100 conjugate (Fig. 2iv) were formed; this is indicated by the protein containing outer aqueous phase stained red, the intermediate oil phase stained green and the inner protein containing water droplets stained red. On formation, the NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions contained a higher proportion of small (water-containing) oil droplets (W1/O) (Fig. 2iv, a) when compared to the (water-containing) oil droplets (W1/O) in the W1/O/W2 double emulsions stabilized by NaCN and NaCN–Md40 conjugate. Storage of these emulsions for 7 days at 45 °C resulted in a large increase in the size of the (water-containing) oil droplets (W1/O) in the W1/O/W2 double emulsion stabilized by NaCN, a somewhat smaller increase in the size of these droplets in the W1/O/W2 double emulsion stabilized by the NaCN–Md40 conjugate, while there was minimal change observed in the size of these droplets in the W1/O/W2 double emulsion stabilized by NaCN–Md100 conjugate. These CSLM images support the result observed for the droplet size distributions (Fig. 1). Both conjugate stabilized W1/O/W2 double emulsions contained (water-containing) oil droplets that remained smaller in size and were also more uniformly distributed in the outer aqueous phase following storage of the emulsions when compared to the NaCN stabilized double emulsions, indicating that these conjugates confer improved flocculation and coalescence stability on W1/O/W2 double emulsions. 3.2.3. Viscosity of W1/O/W2 double emulsions The viscosity of an emulsion is an important characteristic since it influences the rate of creaming, the physical shelf-life of the product and the organoleptic properties of the product (McClements, 1999); viscosity changes on storage may also be used to indicate the type of emulsion instability that occurs on storage (Dickinson, Golding, & Povey, 1997; McClements, 1999). Fig. 3 shows typical viscosity profiles (viscosity versus shear rate) of the W1/O/W2 double emulsions after preparation and following storage of the emulsion for up to 7 days at 45 °C. Following preparation, the NaCN stabilized W1/O/W2 double emulsion had minimal shear thinning behaviour and had a mean constant shear viscosity of 58 mPa s. The NaCN–Md100 conjugate stabilized W1/O/W2 double emulsion also had minimal shear thinning behaviour and had a slightly higher mean constant shear viscosity of 62 mPa s. The NaCN–Md40 conjugate stabilized double emulsion had the highest shear thinning behaviour on formation, and had a slightly higher typical mean constant shear viscosity of 85 mPa s, which may be attributed to the higher water binding of the NaCN– Md40 conjugate and of the non-conjugated Md40 in the continuous phase of the emulsion; this enhanced water binding is attributed to the longer oligosaccharide chain length of the Md40 in comparison to the Md100. On storage of the emulsions under accelerated shelf life testing conditions at 45 °C for up to 7 days, the viscosity profile of the NaCN stabilized W1/O/W2 double emulsion became more shear

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(a) d43 (♦) = 2.46 ± 0.36µm NaCN-Md40 conjugate (Δ) = 3.20 ± 0.19µm NaCN-Md100 conjugate (o) = 1.92 ± 0.17µm

14

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Droplet size (µm) Fig. 1. Typical droplet size distribution and mean droplet size (d43) of W1/O/W2 double emulsions stabilized with NaCN (), NaCN–Md40 conjugate (D) and NaCN–Md100 conjugate (s), immediately after manufacture (a) and after 7 days storage at 45 °C (b).

Fig. 2. Confocal Scanning Laser Microscopy (CSLM) images of the water-in-oil (W1/O) emulsion (i), and of the water-in-oil-in-water (W1/O/W2) double emulsions stabilized with NaCN (ii), NaCN–Md40 conjugate (iii) and NaCN–Md100 conjugate (iv) as the outer aqueous phase (W2) emulsifier on emulsion formation (a) and following storage for 7 days at 45 °C (b). Emulsions were stained with Nile blue and the images show the aqueous protein phases in red and the oil phase in green. Scale bar: 10 lm.

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dependant than that of the NaCN–Md40 conjugate stabilized W1/ O/W2 double emulsion, while the NaCN–Md100 conjugate stabilized double emulsion exhibited minimal, if any change in shear dependent viscosity (Fig. 3). In addition, the mean constant shear viscosity of both of the NaCN–Md conjugate stabilized W1/O/W2 double emulsions remained relatively unchanged over the 7 days of storage at 45 °C (Fig. 4), while the mean constant shear viscosity

of NaCN stabilized W1/O/W2 double emulsion increased from 58 mPa s to 93 mPa s. The higher shear dependence and increase in mean constant shear viscosity of the NaCN stabilized W1/O/W2 double emulsions on storage of the emulsion for up to 7 days at 45 °C may be attributed to droplet flocculation and coalescence, while the low shear dependence of viscosity of the NaCN–Md conjugate stabilized W1/O/W2 double emulsions indi-

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7

Time (Days) Fig. 4. The mean constant shear viscosity of W1/O/W2 double emulsions stabilized with NaCN (); NaCN–Md40 conjugate (D) and NaCN–Md100 conjugate (s), on the day of manufacture (day 0) and on storage at 45 °C for up to 7 days.

served with increasing storage time for all of the emulsions (Fig. 5). Over the 7 day storage period the amount of Vitamin B12 encapsulated in the inner aqueous phase (W1) was significantly (p < 0.05) reduced from 16.6% to 10.1% (total reduction of 39.2%), from 22.4% to 14.7% (total reduction of 34.3%) and from 27.4% to 19.8% (total reduction of 27.7%) for NaCN, NaCN–Md40 and NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions, respectively. Overall, these results show that the double emulsions stabilized by both NaCN–Md40 and NaCN–Md100 conjugates maintained improved inner aqueous phase (W1) encapsulation on storage at 45 °C, in comparison to the NaCN stabilized double emulsion. Fechner et al. (2007) prepared NaCN stabilized neutral pH W1/ O/W2 double emulsions using a similar method to that used in this study; using Vitamin B12 as an inner aqueous phase marker they reported encapsulation efficiencies of 13–19%, which are generally similar values to those reported in this study for NaCN stabilized double emulsions; they reported that conjugation of NaCN with dextrans resulted in slight improvements in the encapsulation efficiencies of neutral pH W1/O/W2 double emulsions when compared

cate that no droplet flocculation occurred on storage of the emulsions; these result are supported by the droplet size distributions and CSLM images (Figs. 1 and 2). 3.3. Encapsulation efficiency and encapsulation stability of W1/O/W2 double emulsions On centrifugation of the freshly prepared NaCN, NaCN–Md40 and NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions, 16.6 ± 1.1%, 22.4 ± 0.5% and 27.4 ± 0.2% of the Vitamin B12 initially added to the inner aqueous phase (W1) remained encapsulated (encapsulation efficiency) in the inner aqueous phase (W1) of the emulsion, respectively (Fig. 5). These results show that both conjugate stabilized W1/O/W2 double emulsions exhibited significantly (p < 0.05) improved encapsulation efficiency when compared to the NaCN stabilized W1/O/W2 double emulsion. On storage of the NaCN, NaCN–Md40 and NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions at 45 °C, a significant reduction (p < 0.05) in the amount of Vitamin B12 encapsulated (encapsulation stability) in the inner aqueous phase (W1) was ob30

cw

Encapsulation (%)

25 20

cx

bw

cy cz bx

aw

by ax

15

bz ay ay

10 5 0 0

2

4

7

Days of storage at 45oC Fig. 5. The percentage of total Vitamin B12 marker added to the inner aqueous phase (W1) of the double emulsion that remains encapsulated in the inner aqueous phase (W1) of water-in-oil-in-water (W1/O/W2) double emulsions stabilized with NaCN (h), NaCN–Md40 conjugate ( ) and NaCN–Md100 conjugate ( ) on emulsion formation (encapsulation efficiency, Day 0) and following storage under accelerated shelf life testing conditions of 45 °C for up to 7 days (encapsulation stability). (a–c) Values with a different letter at the same storage day are significantly different (p < 0.05). (w–z) Values with a different letter across columns (storage days) are significantly different (p < 0.05).

J. O’Regan, D.M. Mulvihill / Food Research International 43 (2010) 224–231

to NaCN stabilized double emulsions. The results of Fechner et al. (2007) and our results support the suggestion of Ueda and Matsumoto (1991) that Vitamin B12 is sufficiently small that it may rapidly diffuse from the inner aqueous phase (W1) across the intermediate oil phase into the outer aqueous phase (W2) on preparation and storage of the W1/O/W2 double emulsions; hence, there is low percentage encapsulation of Vitamin B12 in double emulsions. Fechner et al. (2007) also reported that conjugates improved the encapsulation efficiency of Vitamin B12 compared to NaCN in double emulsions acidified to pH 4.0; this was attributed to the improved solubility of the conjugates at acidic pH compared to the native protein. Improved solubility at acidic pH was previously reported for the conjugates used in this study (O’Regan and Mulvihill, 2009a), which suggests that they may also enhance the encapsulation properties of W1/O/W2 double emulsions under acidic conditions. 4. Conclusions NaCN–Md40 and NaCN–Md100 conjugate stabilized W1/O/W2 double emulsions had improved general stability when compared to NaCN stabilized W1/O/W2 double emulsions on storage for 7 days at 45 °C. Conjugate stabilized double emulsions exhibited less flocculation/coalescence (increase in droplet diameter), less change in constant shear viscosity and less change in shear dependence of viscosity than caseinate stabilized double emulsions. Double emulsions stabilized with NaCN–Md40 and NaCN–Md100 conjugates had better inner phase encapsulation efficiency and encapsulation stability of Vitamin B12 than the NaCN stabilized double emulsion. Overall, these results indicate the potential of NaCN–Md conjugates to produce more stable double emulsions than NaCN. References Akhtar, M., & Dickinson, E. (2003). Emulsifying properties of whey protein–dextrin conjugates at low pH and different salt concentrations. Colloids and Surfaces B: Biointerfaces, 31, 125–132. Allouche, J., Tyrode, E., Sadtler, V., Choplin, L., & Salager, J. L. (2003). Single- and twostep emulsification to prepare a persistent multiple emulsions with a surfactant–polymer mixture. Industrial Engineering Chemical Research, 42, 2988–3982. AOAC (1995). Official methods of analysis (16th ed., pp. 1–75). Arlington, VA, USA: Association of Official Analytical Chemists. Appelqvist, I. A. M., Golding, M., Vreeker, R., & Zuidam, J. (2007). Encapsulation and controlled release technologies in food systems (pp. 41–81). Benichou, A., Aserin, A., & Garti, N. (2004). Double emulsions stabilized with hybrids of natural polymers for entrapment and slow release of active matters. Advances in Colloids and Interface Science, 108–109, 29–41. Benichou, A., Aserin, A., & Garti, N. (2007). W/O/W double emulsions stabilized with WPI-polysaccharide complexes. Colloids and Surfaces A. Physicochemical and Engineering Aspects, 294, 20–32. Benna-Zayani, M., Kbir-Ariguib, N., Trabelsi-Ayadi, M., & Grossiord, J.-L. (2008). Stabilisation of W/O/W double emulsion by polysaccharides as weak gels. Colloids and Surfaces A. Physicochemical and Engineering Aspects, 316, 46–54. Chevalier, F., Chobert, J. M., Popineau, Y., Nicolas, M. G., & Haertlé, T. (2001). Improvement of functional properties of b-lactoglobulin glycated through the Maillard reaction is related to the nature of the sugar. International Dairy Journal, 11, 145–152. Dalgleish, D. G. (1997). Structure-function relationships of caseins. In S. Damodaran & A. Paraf (Eds.), Food proteins and their applications (pp. 199–223). New York: Marcel Dekker.

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