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Physicochemical properties and riboflavin encapsulation in double emulsions with different lipid sources
LWT - Food Science and Technology 59 (2014) 621e628
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Physicochemical properties and riboflavin encapsulation in double emulsions with different lipid sources nez-Colmenero Ricard Bou*, Susana Cofrades, Francisco Jime Institute of Food Science, Technology, and Nutrition (ICTAN-CSIC), Jose Antonio Novais, 10, 28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2014 Received in revised form 7 May 2014 Accepted 10 June 2014 Available online 25 June 2014
This paper examines the influence of the type of lipid source (chia oil, sunflower oil, olive oil or rendered pork backfat) on physicochemical properties and riboflavin encapsulation in food-grade W1/O/W2 double emulsions (DE) for use as functional healthier-fat food ingredients. DEs with encapsulated riboflavin were subjected to conventional thermal treatment (70 C for 30 min) and storage at 4 C for 8 days to determine their influence on oil droplet particle size characteristics, viscosity, dynamic rheological properties, physical stability and encapsulation efficiency. The thermal treatment caused minimal changes in these parameters. DEs containing rendered pork backfat (DEsRPF) collapsed after 3 days of storage at 4 C, thus limiting their useful life. For that reason samples of this DEsRPF were stored at room temperature only and proved stable throughout storage in those conditions. Riboflavin was efficiently encapsulated, although the DEs containing chia oil (DEsCO) were the most efficient at the start. However, these DEs released riboflavin progressively during storage at 4 C. After 8 days' storage at 4 C, DEsRPF stored at room temperature had encapsulated riboflavin more efficiently than DEs containing oil sources. Overall, DEs were stable to environmental stresses typically occurring in the food industry. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Double emulsion Encapsulation Riboflavin Lipid phase Functional ingredient
1. Introduction Multiple emulsions are multi-compartmentalized systems where the globules of the dispersed phase themselves contain even smaller dispersed droplets (Garti, 1997). The two major types of multiple emulsions are water-in-oil-in-water (W/O/W) and oil-inwater-in-oil (O/W/O) double emulsions (DE). These emulsions have a number of interesting properties such as the ability to trap and protect various substances as well as being able to control their release from the inner phase to the outer phase. DEs offer some advantages for food applications, since this has been found to be a potentially useful strategy to enclose nutritional and bioactive compounds. These emulsions could be used as food ingredients in neznew food systems such as healthier and functional foods (Jime Colmenero, 2013). Riboflavin deficiency has been documented in industrialized and developing nations and is usually associated with some severe diseases (Food Nutrition Board, 1998; Powers, 2003). Therefore, the development of foods fortified with riboflavin is a suitable strategy
* Corresponding author. Tel.: þ34 91 549 2300; fax: þ34 91 549 3627. E-mail addresses: [email protected], [email protected] (R. Bou). http://dx.doi.org/10.1016/j.lwt.2014.06.044 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
to protect certain groups of people at risk of riboflavin deficiency and to prevent and treat different disorders (Condo, Posar, Arbizzani, & Parmeggiani, 2009; Horvath, 2012). In this context, the development of meat products enriched with riboflavin would be particularly useful in combating the high prevalence of moderate riboflavin and iron deficiencies in certain population groups (de Benoist, McLean, Egli, & Cogswell, 2008). However, the development of riboflavin-enriched foods is not easy because of riboflavin's intrinsic yellowish colour and its liability to promote photosensitized reactions (Cardoso, Libardi, & Skibsted, 2012). This means that it needs to be encapsulated, which can be achieved by means of DE technology. Because of their health implications, lipids are among the bioactive components (functional ingredients) that have received most attention. In this respect, DEs can be used to modify qualitative and quantitative aspects of the lipid material in foods, improving their content through two main approaches: by reducing the fat content and by providing healthier fatty acid (FA) profiles (e.g. n-3 FA). However, there has been very little use of DEs as fat replacers (food ingredients) in reformulation processes to produce low-fat (low-energy) foods. The majority of cases where DEs have been used as food ingredients involve various novel reduced-fat dairy products in which milk fat has been replaced
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with a W1/O/W2 DE (Lobato-Calleros, Recillas-Mota, EspinosaSolares, Alvarez-Ramirez, & Vernon-Carter, 2009; Marquez & Wagner, 2010). Preliminary experiments which have also been performed to develop food-grade DEs as ingredients to reduce the animal fat content in meat or other food reformulation processes have reported that it is feasible to use these DEs as animal fat renez-Colmenero, placers (Cofrades, Antoniou, Solas, Herrero, & Jime 2013). Besides the nutritional and health implications, the nature of the oil phase used in the formation of the primary emulsion influences various properties such as DE stability and encapsulation efficiency. The stability of W1/O and W1/O/W2 emulsions is strongly affected by the composition and physicochemical properties of the oil phase. Although most available vegetable oils usually have relatively high viscosity and higher water solubility than mineral oils, they can form stable multiple emulsions in association with large lipophilic and hydrophilic molecular emulsifiers (Su, Flanagan, Hemar, & Singh, 2006; Su, Flanagan, & Singh, 2008). The oil phase serves as a sort of membrane that separates the two parts of the aqueous phase, influencing the ability of the materials to transport between the inner and the outer phase (Lutz, Aserin, Wicker, & Garti, 2009). The nature of the oil also determines the droplet size, interfacial tension and stability, which in turn may affect the diffusion rates of hydrophilic species retained within the inner aqueous phase through the oil phase (Bonnet et al., 2009; Garti, 1997; Weiss & Muschiolik, 2007). Although kinetic release of compounds entrapped in the inner aqueous phase of the DE depends on the bioactive compound, the release kinetics of compounds such as L-tryptophan (Weiss, Scherze, & Muschiolik, 2005), sodium ascorbate (Lutz et al., 2009) or magnesium (Bonnet et al., 2009) has also been found to be dependent on the type of oil used in the W1/O/W2 emulsion. The objective of this study was therefore to examine the influence of the lipid source used as a lipid phase of the primary emulsion on riboflavin encapsulation and physicochemical properties of food-grade W1/O/W2 emulsions. To that end, various DEs were prepared with different vegetable oils in order to be able to both reduce and improve the FA profile of foods when used as food ingredients. In addition, animal fat was used to fabricate DEs as an alternative strategy for reducing fat content, thus avoiding the use of fat sources that are not habitual in some food matrices.
2.2. Preparation of double emulsions (DEs) A two stage procedure was used to prepare stable DEs, as reported elsewhere (Cofrades et al., 2013) with minor modifications. The inner (W1) phase consisted of a 5.84 g/L aqueous solution of NaCl plus 0.03 g/100 mL riboflavin and was protected from light. The outer (W2) phase was prepared by dispersing 5.84 g/L NaCl plus 0.5 g/100 mL sodium caseinate in distilled water at room temperature until fully dissolved. Thereafter, 0.02 g/100 mL sodium azide was added to the outer phase to prevent microbial growth and allow us to study their stability. The pH of the inner and outer aqueous phases was in the range 5.7e6.5 at all times. The lipid phase (O) consisted of chia, sunflower or olive oil, or RPF (94 g/ 100 g), plus PGPR as the lipophilic surfactant (6 g/100 g). Phases were mixed for 15 min at 60 C in a Thermomix TM-31 food processor (Vorwerk, Germany) at setting 5. Four primary coarse emulsions (W1/O) were prepared by dropwise addition of the inner (W1) aqueous phase (20 g/100 g) to each of the four lipid phases (80 g/100 g) in the Thermomix food processor set at 60 C for 15 min at setting 6. Each primary coarse emulsion was passed twice through a two-stage high pressure homogenizer at 55,000/7,000 kPa (Panda Plus 1000, GEA NiroSoavi, Parma, Italy). The resulting primary fine emulsions (W1/O) were then allowed to cool at room temperature before preparation of the DEs. These were prepared by gradually adding each W1/O fine emulsion (40 g/100 g) to the outer (W2) aqueous phase (60 g/100 g) in the Thermomix food processor set at 37 C and at setting 3. The resulting coarse W1/O/W2 emulsions were passed twice through a two-stage high pressure homogenizer (Panda Plus 1000) at 15,000/3,000 KPa to obtain the final DEs containing chia, sunflower or olive oil or RPF (DEsCO, DEsSO, DEsOO, DEsRPF respectively). These DEs are expected to be used as ingredients for the intended food application immediately or stored for as short a time as possible for a number of reasons (economics, organisation of production tasks, safety, etc.). 2.3. Thermal treatment and storage
2. Material and methods
Immediately after DE preparation, 10 mL aliquots were added to graduated plastic tubes. Half of the tubes were immediately stored at 4 C. The other half were heated for 30 min in a water bath at 70 C and stored at 4 C after cooling. Additionally, heated and unheated DEs prepared with RPF were stored at room temperature (approx. 22 C). All samples were analysed at 1, 3, 6 and 8 days of storage unless otherwise specified.
2.1. Materials
2.4. Physical stability
In ascending order of FA unsaturation, unrefined chia vegetable oil (Primaria Premium Raw Materials, S.L; Valencia, Spain), refined sunflower oil (Koipesol Semillas S.A.; Sevilla, Spain) and tara S.A.; Madrid, virgin olive oil (Carbonell Virgen Extra, SOS Cue Spain) were used in the formation of the lipid phase of W1/O/W2 emulsions because their FA profiles (oils rich in n-3, n-6 and n-9 series respectively) are healthier than those of animal fats (rich in saturated FAs). Approximately 600 g of ground Iberian pork backfat purchased in a local market was heated up to 70 C in a water bath and then filtered through cheesecloth to obtain rendered pork backfat (RPF), for use as the lipid phase in the formation of reduced fat ingredients. Polyglycerol polyricinoleate (PGPR; SUGIN 476/M) was purchased from Cargill S.L.U. (Martorell, Spain). Sodium caseinate was from DMV (Excellion EM 7, DMV Campina B.V.; Veghel, The Netherlands). Sodium chloride was from Panreac (Panreac Química, S.A.; Barcelona, Spain). Riboflavin and sodium azide were from Sigma-Aldrich (Madrid, Spain).
Gravitational separation (creaming) of heated and unheated DEs was recorded in triplicate over storage in terms of phase separation and expressed as percentage of initial sample height. 2.5. Microscopy Light microscopy was used to examine emulsion morphology. Samples were diluted 8 times with 5.84 g/L NaCl, placed on microscope slides and carefully covered with a cover slip. The DE microstructure was observed using a Reichert microscope (Munich, Germany) at 40 and 100 magnifications. Measurements in heated and unheated DEs were made at days 1 and 8 of storage. 2.6. Particle size characteristics The particle size and distribution of oil droplets in the heated and unheated DEs was determined immediately, after 10-fold
R. Bou et al. / LWT - Food Science and Technology 59 (2014) 621e628
dilution with 5.84 g/L NaCl, with a Malvern Mastersizer S laser diffraction particle size analyser (Malvern Instrument Ltd, Worcestershire, UK) equipped with a He-Ne laser (l ¼ 633 nm). The measurement range was 0.05e900 mm. Obscuration was in the range of 8e15%. Particle size calculations were based on the Mie Scattering theory. Surface average diameter (d32) was measured immediately after addition to the dispersion unit. Results as reported were averages of at least three measurements.
EE ð%Þ ¼ 100
CW2 100 CW1
623
where Cw2 is the concentration of riboflavin determined in the outer aqueous phase, and Cw1 is the concentration of riboflavin considering the amount of the vitamin initially added to the inner aqueous phase and the equivalent dilution produced by mass transfer of water from the inner phase to the aqueous phase. All samples were analysed in triplicate.
2.7. Viscosity 2.10. Statistical analysis The viscosity of the different lipid phases used for the production of DEs and that of the resulting heated and unheated DEs containing vegetable oils was determined at 4 C, whereas the viscosity of RPF and DEsRPF was determined at two temperatures (4 and 25 C). These measurements were done in a CP4/40 coneplate cell (4 angle and 40 mm diameter) with a 150 mm gap on a Bohlin CVO-100 rheometer (Bohlin Instruments Ltd., Gloucestershire, UK) at a constant shear stress of 0.5 s1. Analyses were done at the beginning of storage. Results, expressed as Pa$s, were averages of five determinations. 2.8. Dynamic rheological properties Dynamic rheological experiments were conducted on unheated DEs using a Bohlin CVO-100 controlled-stress rheometer (Bohlin Instruments Ltd., Gloucestershire, UK). The measurement system was a cone plate geometry CP4/40 (4 angle and 40 mm diameter) with a 150 mm gap in all samples. Samples were allowed to relax for 5 min before rheological measurements such as equilibration time after loading the sample on the sensor system. Temperature was controlled with a Peltier Plate system (40 to þ180 C; Bohlin Instruments, Gloucestershire, UK). The linear viscoelastic region was determined for each sample through stress sweeps at 0.1 Hz. After that, a dynamic frequency sweep was conducted by applying a constant pre-determined stress within the linear region, over a frequency range between 0.05 and 1 Hz for each sample. The measurements were made at 25 C. Parameters including elastic modulus (G0 ), viscous modulus (G00 ) and phase angle (d) were derived using the analysis program software. Analyses were done at the beginning of storage. Results were averages of at least three measurements. 2.9. Determination of riboflavin encapsulation efficiency and stability Encapsulation efficiency can be defined as the amount of the bioactive compound which remains entrapped in the inner aqueous phase (W1). The release of riboflavin was determined after each storage period (encapsulation stability) by 4-fold dilution of the DE in a 5.84 g/L aqueous solution of NaCl. Samples were then centrifuged at 2,500 g for 30 min (Heraeus multifuge 3L-R DJB Labcare Ltd.; Buckinghamshire, UK) to separate the fat globules from the outer aqueous phase. These centrifugation conditions did not change d32 after centrifugation. Four mL of the lower aqueous phase was taken out and mixed with 2 mL of 33 g/100 mL trichloroacetic acid. Samples were then filtered through Whatman No. 1 filter paper. The absorbance of the filtrate was measured at 382 nm using a spectrophotometer (Shimadzu UV-1800; Japan). Concentrations were determined from a riboflavin standard curve. The encapsulation efficiency (EE) was calculated with respect to the amount of riboflavin added to the inner aqueous phase using the following equation (Sapei, Naqvi, & Rousseau, 2012):
The effects of the type of lipid phase used (chia, sunflower and olive oils stored at 4 C and RPF stored at room temperature) in the fabrication of the DEs, the thermal treatment (where applicable) and chilling storage of these DEs, on mean particle size, viscosity, physical stability and encapsulation efficiency were analysed using a multifactor ANOVA. Because RPF is solid/semisolid, only the effect of vegetable oils on lipid phase viscosity was included in the statistical analysis. The effects of storage temperature and thermal treatment on the same parameters were considered for DEsRPF stored for 1 day at either 4 C or at room temperature. Interactions between two factors were also considered. A P-value of less than 0.05 was considered significant. Tukey-HSD tests were used to identify statistically significant differences except for viscosity of DEs, for which the Tamhane test was used. Statistical analyses were carried out using IBM SPSS Statistics 21 software. 3. Results and discussion These emulsions must be considered in the context of their practical use. In particular, they are designed for use as ingredients in meat product reformulation processes. Accordingly, all DEs were formulated to contain 2.4 mg of riboflavin per 100 g of DE, since addition to meat systems at levels ranging from 10 to 15 g/100 g is realistic (Cofrades et al., 2013). This means 0.24e0.36 mg of riboflavin per 100 g of food (meat product), which covers 17e25% of the Recommended Dietary Allowances for pregnancy (1.4 mg) (Food Nutrition Board, 1998). As formulated, these DEs were expected to have similar fat contents (32 g/100 g). In meat reformulation processes, the use of RPF in the fabrication of DEs makes it possible to develop novel ingredients to reduce the animal fat content while maintaining sensory properties. Other lipid sources with positive health implications can be used in the preparation of DEs to develop novel ingredients for healthier, low-energy foods. 3.1. Physical stability These DEs have to be prepared beforehand and used as one of a number of ingredients in meat product formulations. Like other perishable meat and non-meat ingredients, they may require cold storage, which needs to be minimized, to prevent microbial growth and maintain stability before they are used as ingredients. These DEs can be heated once they have been added to meat systems to produce cooked meat products, and we therefore also addressed their stability to conventional thermal treatments. All heated and unheated DEs had a homogenous milky white appearance after preparation. As they are for use as fat replacers, this milky appearance is not a problem. Moreover, this appearance was maintained during storage at 4 C for 8 days in those DEs containing vegetable oils. However, heated and unheated DEsRPF samples collapsed and changed to a stiff and creamy homogenous phase after 48 h of cold storage, which limits their utility as food ingredients to that period of time. When DEsRPF was stored at
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room temperature it was found to be stable over time. This suggests that the loss of the initial structure is likely due to the formation of large crystals, causing disruption of the emulsion and concomitant formation of a fat crystal network during storage at 4 C. In an earlier experiment with DEs containing pork lard in the lipid phase and sodium caseinate in the outer water phase, storage at 4 C nezcaused the formation of some clumps (Bou, Cofrades, & Jime Colmenero, 2014). Therefore, it seems that chilling storage of DEs containing fats causes some instability problems, probably due to the high melting point of the lipid phases. The fact that the initial structure of DEsRPFs was maintained within the first 48 h of storage at 4 C is enough to warrant their use in the meat industry. Nevertheless, we decided to compare DEsRPF stored at room temperature with DEs containing vegetable oils stored at 4 C. At the starting time, samples DEsSO and DEsOO showed no signs of creaming (Table 1). In contrast, DEsCO and DEsRPF (this last stored at room temperature) showed little phase separation. At the end of the storage period, phase separation was higher (21.3%) in DEsRPF stored at room temperature, while creaming varied in DE containing vegetable oils (Table 1). Overall, the physical stability of the DEs containing vegetable oils was acceptable and in good agreement with other very similar DEs containing olive oils (Cofrades et al., 2013). Of the DEs containing vegetable oils, the phase separation was 14.0% in DEsOO, decreasing with increasing unsaturation in the lipid phase. Creaming was most intense in DEsRPF, but these samples were stored at room temperature. However, their stability was not very different from that of DEs containing pork lard and stored at 4 C. Bou et al. (2014) compared the stability of DEs containing olive oil and pork lard and reported that, under storage at 4 C, DEs containing olive oil were less stable than DEs containing pork lard (89% and 97%, for olive oil and pork lard respectively). Although temperature is critical for DE stability, it is reasonable to assume that DEsRPFs are quite stable to creaming. Only DEsCO and DEsRPF samples were affected by thermal treatment (data not shown). When DEsCO and DEsRPF (this last stored at room temperature) were heated, phase separation was greater than in the unheated DEs (5.7 vs 7.4% and 11.9 vs 12.9% for unheated versus heated DEsCO and DEsRPF respectively). 3.2. Morphology Fig. 1 shows the microscopic structure of unheated DEsOO, which is representative of the rest. It can be observed that a large
Fig. 1. Light microscopy images of unheated W1/O/W2 emulsions containing olive oil after 8 days of storage at 4 C. Scale bars represent 10 mm at 40 and 100 magnifications.
amount of small oil globules coexist with only some scarce bigger droplets. Irrespective of the size, all droplets have the characteristic structure of a DE, consisting in small water droplets located inside an oil globule; however, because they are small, this structure can be more easily observed in these larger droplets at 100 magnification (Fig. 1 insert). It is important to note that this compartmentalized structure was also observed in all DEsRPFs. However, the structure collapsed in the course of storage at 4 C (not shown). In an earlier experiment, it was also observed that the visual appearance and overall microscopic structure of DEs containing olive oil did not differ greatly from those containing pork lard (Bou et al., 2014). The different lipid sources used to fabricate DEs therefore seemed to have very little effect on their morphology. DEsRPF was unstable after 8 days of storage at 4 C and, as noted earlier, the appearance changed from milky to creamy. However, DEsRPF stored at room temperature and DEsCO, DEsSO and DEsOO stored at 4 C were apparently unchanged after 8 days of storage (not shown). The DEs were heated at 70 C for 30 min to mimic a conventional thermal treatment in the food industry (e.g. meat processing) and thereafter stored under the same conditions as unheated DEs. The compartmentalized structure was unaffected by heating and there
Table 1 Particle size (d32), physical stability (creaming) and encapsulation stability of W1/O/W2 emulsions as affected by the lipid phase.a Day
DEsCO
DEsSO
DEsOO
DEsRPF
Stored at 4 C Particle size (d32; mm)
Physical stability (%)
Encapsulation stability (%)
1 3 6 8 1 3 6 8 1 3 6 8
1.58aX 1.24aX 2.02bX 2.10b 0.7aY 2.8abY 5.2abX 6.8bV 85.4dZ 78.6bX 80.9cY 76.6aX
± ± ± ± ± ± ± ± ± ± ± ±
0.27 0.38 0.10 0.11 0.51 2.86 3.86 3.87 0.79 0.25 0.94 1.06
Stored at 22 C 2.32abY ± 2.49bZ ± 2.28aZ ± 2.20a ± 0aX 2.1bX ± 4.8cX ± 11.2dX ± 81.6bXY ± 82.5bY ± 81.4abY ± 80.4aY ±
0.06 0.13 0.19 0.09 0.20 0.41 0.75 0.60 0.40 0.72 1.46
2.20Y ± 2.16Y ± 2.22YZ ± 2.10 ± 0aX 1.3bV ± 6.8cY ± 14.0dY ± 80.4bX ± 75.4aV ± 75.2aX ± 76.7aX ±
0.17 0.29 0.10 0.19 0.41 0.75 0.89 0.55 0.75 1.37 1.63
2.18Y 2.07Y 2.05XY 2.06 1.12aZ 8.0bZ 19.2cZ 21.3dZ 82.1aY 85.3cZ 84.3bcZ 83.2abZ
± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.10 0.05 0.11 0.20 0.63 0.75 1.21 1.94 0.64 0.79 0.72
Means within the same column without a common letter (aed) are significantly different (P 0.05). Means within the same row without a common letter (VeZ) are significantly different (P 0.05). a DEsCO, DEsSO, DEsOO and DEsRPF stand for double emulsions containing chia oil, sunflower oil, olive oil and rendered pork backfat respectively. Results for DEsRPF stored at 4 C are not shown because this emulsion was not stable throughout storage. Values given in this table correspond to least-square means ± standard deviation obtained from MANOVA (n ¼ 48).
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were no appreciable changes in their morphology (results not shown). Therefore, all these DEs can be used in the development of foods that require some thermal treatment and hence may be suitable for use as low fat ingredients in the processing of many food products. 3.3. Particle size characteristics Regardless of the lipid source and storage time, the DEs displayed unimodal distributions, although with differences in width (Figs. 2 and 3). Unimodal distribution has also been reported in other similar DEs in which PGPR and sodium caseinate were used respectively as lipophilic and hydrophilic emulsifiers (Cofrades et al., 2013; Frasch-Melnik, Spyropoulos, & Norton, 2010). At the start, d32 of DEsSO, DEsOO and DEsRPF were similar, while the droplet diameter of the DE containing the most unsaturated lipid source (DEsCO) was smaller (Table 1 and Fig. 2). These sizes are in agreement with other very similar DEs containing olive oil and pork lard (Bou et al., 2014). Bonnet et al. (2009) compared different lipids (olive oil, rapeseed oil, olein and miglyol) for use in the formulation of DEs. These authors reported similar droplet diameters regardless of the lipid source used. Conversely, other authors found different sizes when examining the effect of different semicrystalline lipids on the average lipid droplet diameter of the resulting DE, although they reported no clear trend and offered no explanation (Weiss et al., 2005). The comparison between DEsRPF stored for 1 day at 4 C and stored at room temperature showed that droplets were larger in the latter case (2.18 vs 2.00 mm for DEs stored at room temperature and at 4 C respectively). Thus, the nature of the lipid used in DEs can influence lipid globule sizes to some extent, but other factors such as viscosity, W1 droplet size and interfacial tension have also been reported to influence particle size and need to be considered to understand and prepare DEs with tailored properties (Weiss & Muschiolik, 2007). DEsSO, DEsOO and DEsRPF (the latter stored at room temperature) showed no increase in oil globule sizes after 8 days of storage (Table 1). In the case of DEsCO, the oil droplet size was unchanged up to 3 days of storage at 4 C but increased over longer storage times (Table 1, Fig. 3). This increase in DEsCO was a factor in the absence of differences between DEs with different lipid sources at the end of the storage period. Chia oil is very rich in linolenic acid (Ayerza & Coates, 2009), and therefore it displayed lower viscosity
Fig. 3. Particle size distribution of W1/O/W2 emulsions containing chia oil after 1 day and 8 days of storage at 4 C.
(0.148 Pa s) because it was more unsaturated (Table 2), which in turn may have contributed to this difference in behaviour. The thermal treatment applied (30 min at 70 C) had no effect on oil droplet distribution or average droplet diameter (global means were 2.06 and 2.09 mm for fresh and heated DEs respectively). And again, the thermal treatment had no effect when DEsRPF samples stored at two different temperatures were compared. Bonnet et al. (2009) studied stability of DEs containing olive oil to thermal treatments by heating them for 20 s at 72 C and 20 s or 5 min at 92 C. As in the present case, the authors reported that the DEs retained the characteristic DE structure and lipid droplet sizes were unaffected. 3.4. Viscosity The influence of the type of lipid on the viscosity of the different lipid phases (containing 6 g/100 g PGPR) used for the formation of primary emulsions is shown in Table 2. As expected, viscosity decreased with increasing unsaturation of the lipid phase. The stability to creaming of DEs stored for 8 days at 4 C seems to be in inverse relation to lipid phase viscosity (Tables 1 and 2). The physical stability of DEs depends on their viscosity because this hinders phase separation. All DEs showed low viscosities, possibly because this is mainly determined by the viscosity of the
Table 2 Viscosity of lipid phases and their respective W1/O/W2 emulsions.a
Fig. 2. Particle size distribution of W1/O/W2 emulsions containing chia oil (DEsCO), sunflower oil (DEsSO), olive oil (DEsOO) and rendered pork backfat (DEsRPF) after 1 day of storage at 4 C.
Lipid used
Temperature at measurement
Lipid phase (Pa$s)
Unheated W1/O/W2 (kPa$s)
Heated W1/O/W2 (kPa$s)
Chia oil Sunflower oil Olive oil RPF RPF
4 C 4 C
0.148 ± 0.0034a 0.174 ± 0.0051b
31.6 ± 0.4cX 24.0 ± 0.7aX
32.8 ± 0.1cY 24.2 ± 0.9aX
4 C 4 C 25 C
0.253 ± 0.0016c 380 ± 9.8 3.6 ± 0.54
26.8 ± 1.4bX 273 ± 68dX 139 ± 36X
26.8 ± 0.1bX 209 ± 79dX 187 ± 24X
Means measured at 4 C and within the same column without a common letter (aed) are significantly different (p 0.05). Means for W1/O/W2 emulsions within the same row without a common letter (XeY) are significantly different (p 0.05). a The lipid phase contains 6% polyglycerol polyricinoleate. Values are means ± standard deviation of lipid phases and W1/O/W2 emulsions measured at the beginning of storage. All emulsions were stored 1 day at 4 C except those containing rendered pork backfat (RPF) which were stored at room temperature (approx. 22 C).
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continuous phase, which was very low (Table 2). The external aqueous phase was the same for all DEs, and so the differences in viscosity could be directly attributed to the different lipid phases. In fact, the viscosities of DEsRPF were higher than those of DEs containing vegetable oils (Table 2). In the case of DEs containing vegetable oils, the viscosities of DEsOO and DEsSO were similar and lower than the rest. Therefore, the low viscosities found in DEsSO and DEsOO could explain the low stability of the DEs to creaming. However, DEsCO, which was the most unsaturated, showed the highest viscosity of all the vegetable oils. There are various possible explanations for these results. On the one hand, the viscosity of DEs has been associated with the particle size and particle size distribution and hence is a factor determining their stability. The fact that the lipid droplets were initially smaller could explain why DEsCO was the most stable to creaming (Tables 1 and 2). Moreover, the narrower the particle size distribution, the greater was the DE's viscosity and stability to creaming. Such a narrow distribution increases the probability of particleeparticle interactions and would cause the DEsCO to be more resistant to gravity, so that the system is more stable to creaming (Fig. 2). On the other hand, those emulsions with a wide droplet size distribution, as in the case of DEsSO (Fig. 2), had more free space for particles to move around, making it easier for the sample to flow, meaning that it was less viscous (Table 2). Alternatively, the higher viscosity of DEsCO could also be explained by the presence of minor compounds such as mucilages (soluble fibre) that can be present in this type of non-refined oil. These mucilages can bind the water of the DE and could thus explain the observed increase in the viscosity of DEsCO. Moreover, the presence and hydration of mucilages as a consequence of the thermal treatment applied may explain why the viscosity of DEsCO increased while the viscosities of the other DE remained unaffected (Table 2). 3.5. Dynamic rheological properties Dynamic oscillatory shear tests were used to characterize the viscoelastic properties of the DEs containing different types of fat (vegetable or animal). The results from the oscillatory shear tests were expressed in terms of the variation of elastic modulus (G0 ), viscous modulus (G00 ) and phase angle (d) as a function of frequency (Hz). The dynamic mechanical spectra of all DEs showed that the viscous modulus (G00 ) values were higher than the corresponding storage modulus (G0 ) values over the entire frequency range, with G0 values too low and irregular for a Power Law to fit. Similar results were found in DEs formulated with olive oil and pork lard, nonpressurized and pressurized at 400 and 600 MPa (Bou et al., 2014). This observation suggests that viscous behaviour predominates over elastic behaviour; all DEs exhibited liquid-like behaviour regardless of the type of oil used. In all samples, G00 increased and the phase angle remained roughly constant when the frequency increased (Fig. 4a and b). Since the phase angle indicates the viscoelasticity index of a sample, this can be interpreted in terms of “solid-like” emulsions (d ~ 0 ) and “liquid-like” emulsions (d ~ 90 ); the predominantly viscous nature of the emulsions was confirmed by the liquid-like aspect and d values of the DE, which ranged from 78 to 85 (Table 3). Ross-Murphy (1995) described changes in and dependency of G00 and G0 of biopolymers with frequency, indicating that a dilute solution typically has a high G00 to G0 ratio, with both moduli showing a strong frequency dependence. When a system shifts from a fluid to a more solid-like system its G0 increases and crosses over G00 . Such a system is described rheologically as a concentrated solution. However, in our systems no changes in the behaviour of G00 and G0 were observed anywhere within the frequency range. Thus, our DEs retained the dilute
Fig. 4. Variation of the viscous modulus (G00 ) and phase angle (d) as a function of frequency (Hz) as determined in the frequency sweep tests (at 25 C) in W1/O/W2 emulsions. A) G00 (Pa) for unheated W1/O/W2 emulsions made with different fat sources; B) d ( ) for unheated W1/O/W2 emulsions made with different fat sources. See Table 1 for abbreviations.
solution structure, without any major conformational changes over the frequency range studied. This behaviour could be related to the high water content in the double emulsions (~70 g/100 g). Similar results were observed by (Lee, Anema, & Klostermeyer, 2004) in processed cheese spread samples with a water content above 54 g/ 100 g, and they found that G00 was greater than G0 over the entire frequency range studied, with no cross-over at low frequencies. These authors reported that small increases in water content resulted in roughly twofold decreases in G0 and G00 ; however, the phase angle increased with increasing moisture. As there was no change in the water content of our samples, there were no changes Table 3 Fitted values for the viscous modulus as determined in the frequency sweep test for unheated W1/O/W2 emulsions formulated with different lipid sources.a Double emulsion
G00 0 (Pa)
DEsCO DEsSO DEsOO DEsRPF
0.066 0.052 0.059 0.087
± ± ± ±
n00 0.001 0.001 0.001 0.001
0.801 1.077 1.022 0.354
d ( ) ± ± ± ±
0.01 0.01 0.01 0.01
85.03 81.55 84.27 83.66
± ± ± ±
3.37 2.44 3.70 3.37
a DEsCO, DEsSO, DEsOO and DEsRPF stand for double emulsions containing chia oil, sunflower oil, olive oil and rendered pork backfat respectively.
R. Bou et al. / LWT - Food Science and Technology 59 (2014) 621e628
in the phase angle (Fig. 4). However, according to Brummer (2006), the mechanical spectrum of a stable emulsion with greater internal strength has a storage modulus, G0 , higher than its viscous modulus, G00 , and both moduli should be almost parallel throughout the observed frequencies, with a slight increase in the slope at high frequencies. In this respect, DEsRPF behaved more like a stable emulsion since it showed a smaller increase with the frequency (
627
contain a relatively large amount of free FAs (acid value 1 g/ 100 g), and these have been reported to migrate and concentrate in the oilewater interfaces, thus changing the properties and affecting the oxidative stability of oil-in-water emulsions (Waraho, Cardenia, Rodriguez-Estrada, McClements, & Decker, 2009). Also, monoacylglycerides and diacylglycerides that are normally present in olive oils have been shown to be good surfactants. It is therefore reasonable to suppose that these minor compounds may play a role in internal interface stability and in turn may affect the encapsulation of bioactive compounds, probably facilitating the transport of entrapped compounds from the inner water phase. The high viscosity of DEsRPF (Table 2) could explain why the initial riboflavin encapsulation efficiency of DEsRPF was unaltered after 8 days of storage at room temperature (Table 1). DEsRPF thus displayed good riboflavin encapsulation stability (83.2%) at the end of the storage period (Table 1). These results are in line with the report by Weiss et al. (2005) that the release of encapsulated tryptophan was reduced by increasing the solid fat content in the oil phase. These findings can be explained by the fact that semicrystalline lipid phases promote not only increased viscosity (Table 2) but also effective encapsulation. On the other hand, storing these DEs at temperatures below or close to the lipid phase melting point can be critical for riboflavin encapsulation. Finally, it is important to note that the thermal treatment had no effect on riboflavin encapsulation (overall least squares means were 80.6% and 80.7% for unheated and heated DEs respectively). Therefore, bioactive compounds can be encapsulated in the inner water phase, but they can also be entrapped with this technology following a thermal treatment. Thus, DEs can be used as ingredients to develop novel functional food products, such as meat products, that require some kind of thermal treatment. 4. Conclusions DEs can be used to develop healthier and functional foods that can be subjected to thermal treatments while still entrapping bioactive compounds such as riboflavin. In this experiment riboflavin was efficiently encapsulated, although this also depended on the nature of the lipid phase used. Of the lipid phases used to prepare DEs, the ones containing chia oil were the most efficient in encapsulating riboflavin. However, during storage at 4 C, the chia oil DE released riboflavin more rapidly than the ones containing sunflower or olive oil. At this storage temperature, DEs containing RPF need to be used within 48 h because of DE breakdown. Nonetheless, these DEs were stable when stored at room temperature for 8 days and under these conditions exhibited the greatest (83.2%) riboflavin encapsulation stability. Acknowledgements R. Bou has been supported by a contract from the JAE-postdoctoral (CSIC) Program. This research has been supported by project AGL 2011-29644-C02-01 from the Spanish Ministry of Science and Innovation. Primaria Premium Raw Materials, S.L. kindly provided the chia oil. The authors wish to thank A. del Hoyo for her help during analyses. References Ayerza, R., & Coates, W. (2009). Influence of environment on growing period and yield, protein, oil and a-linolenic content of three chia (Salvia hispanica L.) selections. Industrial Crops and Products, 30, 321e324. de Benoist, B., McLean, E., Egli, I., & Cogswell, M. (2008). Worldwide prevalence of anaemia 1993e2005. WHO global database on anaemia WHO Library Cataloguing-in-Publication Data http://whqlibdoc.who.int/publications/2008/ 9789241596657_eng.pdf.
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Bonnet, M., Cansell, M., Berkaoui, A., Ropers, M. H., Anton, M., & Leal-Calderon, F. (2009). Release rate profiles of magnesium from multiple W/O/W emulsions. Food Hydrocolloids, 23, 92e101. nez-Colmenero, F. (2014). Influence of high pressure and Bou, R., Cofrades, S., & Jime heating treatments on physical parameters of water-in-oil-in-water emulsions. Innovative Food Science and Emerging Technologies, 23, 1e9. http://dx.doi.org/ 10.1016/j.ifset.201404.001. Brummer, R. (2006). Rheology essentials of cosmetic and food emulsions. Germany: Springer-Verlag. Cardoso, D., Libardi, S., & Skibsted, L. (2012). Riboflavin as a photosensitizer. Effects on human health and food quality. Food & Function, 3, 487e502. nez-Colmenero, F. Cofrades, S., Antoniou, I., Solas, M. T., Herrero, A. M., & Jime (2013). Preparation and impact of multiple (water-in-oil-in-water) emulsions in meat systems. Food Chemistry, 141, 338e346. Condo, M., Posar, A., Arbizzani, A., & Parmeggiani, A. (2009). Riboflavin prophylaxis in pediatric and adolescent migraine. The Journal of Headache and Pain, 10, 361e365. Fechner, A., Knoth, A., Scherze, I., & Muschiolik, G. (2007). Stability and release properties of double-emulsions stabilised by caseinateedextran conjugates. Food Hydrocolloids, 21, 943e952. Food Nutrition Board. (1998). Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington, DC: The National Academies Press. Frasch-Melnik, S., Spyropoulos, F., & Norton, I. T. (2010). W1/O/W2 double emulsions stabilised by fat crystals e formulation, stability and salt release. Journal of Colloid and Interface Science, 350, 178e185. Garti, N. (1997). Double emulsions: scope, limitations and new achievements. Colloids and Surfaces A, Physicochemical and Engineering Aspects, 123e124, 233e246. Horvath, R. (2012). Update on clinical aspects and treatment of selected vitaminresponsive disorders II (riboflavin and CoQ 10). Journal of Inherited Metabolic Disease, 35, 679e687. nez-Colmenero, F. (2013). Potential applications of multiple emulsions in the Jime development of healthy and functional foods. Food Research International, 52, 64e74. Lee, S. K., Anema, S., & Klostermeyer, H. (2004). The influence of moisture content on the rheological properties of processed cheese spreads. International Journal of Food Science & Technology, 39, 763e771.
Li, B., Jiang, Y. F., Liu, F., Chai, Z., Li, Y. Y., & Leng, X. J. (2011). Study of the encapsulation efficiency and controlled release property of whey protein isolateepolysaccharide complexes in W1/O/W2 double emulsions. International Journal of Food Engineering, 7(3). http://dx.doi.org/10.2202/1556e3758.2321. Lobato-Calleros, C., Recillas-Mota, M. T., Espinosa-Solares, T., Alvarez-Ramirez, J., & Vernon-Carter, E. J. (2009). Microstructural and rheological properties of lowfat stirred yoghurts made with skim milk and multiple emulsions. Journal of Texture Studies, 40, 657e675. Lutz, R., Aserin, A., Wicker, L., & Garti, N. (2009). Release of electrolytes from W/O/W double emulsions stabilized by a soluble complex of modified pectin and whey protein isolate. Colloids and Surfaces B, Biointerfaces, 74, 178e185. Marquez, A., & Wagner, J. (2010). Rheology of double (W/O/W) emulsions prepared with soybean milk and fortified with calcium. Journal of Texture Studies, 41, 651e671. Owusu, R. K., Zhu, Q. H., & Dickinson, E. (1992). Controlled release of L-tryptophan and vitamin B2 from model water/oil/water multiple emulsions. Food Hydrocolloids, 6, 443e453. Powers, H. J. (2003). Riboflavin (vitamin B-2) and health. The American Journal of Clinical Nutrition, 77, 1352e6130. Ross-Murphy, S. B. (1995). Rheological characterisation of gels. Journal of Texture Studies, 26, 391e400. Sapei, L., Naqvi, M., & Rousseau, D. (2012). Stability and release properties of double emulsions for food applications. Food Hydrocolloids, 27, 316e323. Su, J., Flanagan, J., Hemar, Y., & Singh, H. (2006). Synergistic effects of polyglycerol ester of polyricinoleic acid and sodium caseinate on the stabilisation of wateroil-water emulsions. Food Hydrocolloids, 20, 261e268. Su, J., Flanagan, J., & Singh, H. (2008). Improving encapsulation efficiency and stability of water-in-oil-in-water emulsions using a modified gum arabic (acacia (sen) SUPER GUM (TM)). Food Hydrocolloids, 22, 112e120. Waraho, T., Cardenia, V., Rodriguez-Estrada, M., McClements, D. J., & Decker, E. (2009). Prooxidant mechanisms of free fatty acids in stripped soybean oil-inwater emulsions. Journal of Agricultural and Food Chemistry, 57, 7112e7117. Weiss, J., & Muschiolik, G. (2007). Factors affecting the droplet size of water-in-oil emulsions (W/O) and the oil globule size in water-in-oil-in-water emulsions (W/O/W). Journal of Dispersion Science and Technology, 28, 703e716. Weiss, J., Scherze, I., & Muschiolik, G. (2005). Polysaccharide gel with multiple emulsion. Food Hydrocolloids, 19, 605e615.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Physicochemical properties and riboflavin encapsulation in double emulsions with different lipid sources nez-Colmenero Ricard Bou*, Susana Cofrades, Francisco Jime Institute of Food Science, Technology, and Nutrition (ICTAN-CSIC), Jose Antonio Novais, 10, 28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2014 Received in revised form 7 May 2014 Accepted 10 June 2014 Available online 25 June 2014
This paper examines the influence of the type of lipid source (chia oil, sunflower oil, olive oil or rendered pork backfat) on physicochemical properties and riboflavin encapsulation in food-grade W1/O/W2 double emulsions (DE) for use as functional healthier-fat food ingredients. DEs with encapsulated riboflavin were subjected to conventional thermal treatment (70 C for 30 min) and storage at 4 C for 8 days to determine their influence on oil droplet particle size characteristics, viscosity, dynamic rheological properties, physical stability and encapsulation efficiency. The thermal treatment caused minimal changes in these parameters. DEs containing rendered pork backfat (DEsRPF) collapsed after 3 days of storage at 4 C, thus limiting their useful life. For that reason samples of this DEsRPF were stored at room temperature only and proved stable throughout storage in those conditions. Riboflavin was efficiently encapsulated, although the DEs containing chia oil (DEsCO) were the most efficient at the start. However, these DEs released riboflavin progressively during storage at 4 C. After 8 days' storage at 4 C, DEsRPF stored at room temperature had encapsulated riboflavin more efficiently than DEs containing oil sources. Overall, DEs were stable to environmental stresses typically occurring in the food industry. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Double emulsion Encapsulation Riboflavin Lipid phase Functional ingredient
1. Introduction Multiple emulsions are multi-compartmentalized systems where the globules of the dispersed phase themselves contain even smaller dispersed droplets (Garti, 1997). The two major types of multiple emulsions are water-in-oil-in-water (W/O/W) and oil-inwater-in-oil (O/W/O) double emulsions (DE). These emulsions have a number of interesting properties such as the ability to trap and protect various substances as well as being able to control their release from the inner phase to the outer phase. DEs offer some advantages for food applications, since this has been found to be a potentially useful strategy to enclose nutritional and bioactive compounds. These emulsions could be used as food ingredients in neznew food systems such as healthier and functional foods (Jime Colmenero, 2013). Riboflavin deficiency has been documented in industrialized and developing nations and is usually associated with some severe diseases (Food Nutrition Board, 1998; Powers, 2003). Therefore, the development of foods fortified with riboflavin is a suitable strategy
* Corresponding author. Tel.: þ34 91 549 2300; fax: þ34 91 549 3627. E-mail addresses: [email protected], [email protected] (R. Bou). http://dx.doi.org/10.1016/j.lwt.2014.06.044 0023-6438/© 2014 Elsevier Ltd. All rights reserved.
to protect certain groups of people at risk of riboflavin deficiency and to prevent and treat different disorders (Condo, Posar, Arbizzani, & Parmeggiani, 2009; Horvath, 2012). In this context, the development of meat products enriched with riboflavin would be particularly useful in combating the high prevalence of moderate riboflavin and iron deficiencies in certain population groups (de Benoist, McLean, Egli, & Cogswell, 2008). However, the development of riboflavin-enriched foods is not easy because of riboflavin's intrinsic yellowish colour and its liability to promote photosensitized reactions (Cardoso, Libardi, & Skibsted, 2012). This means that it needs to be encapsulated, which can be achieved by means of DE technology. Because of their health implications, lipids are among the bioactive components (functional ingredients) that have received most attention. In this respect, DEs can be used to modify qualitative and quantitative aspects of the lipid material in foods, improving their content through two main approaches: by reducing the fat content and by providing healthier fatty acid (FA) profiles (e.g. n-3 FA). However, there has been very little use of DEs as fat replacers (food ingredients) in reformulation processes to produce low-fat (low-energy) foods. The majority of cases where DEs have been used as food ingredients involve various novel reduced-fat dairy products in which milk fat has been replaced
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with a W1/O/W2 DE (Lobato-Calleros, Recillas-Mota, EspinosaSolares, Alvarez-Ramirez, & Vernon-Carter, 2009; Marquez & Wagner, 2010). Preliminary experiments which have also been performed to develop food-grade DEs as ingredients to reduce the animal fat content in meat or other food reformulation processes have reported that it is feasible to use these DEs as animal fat renez-Colmenero, placers (Cofrades, Antoniou, Solas, Herrero, & Jime 2013). Besides the nutritional and health implications, the nature of the oil phase used in the formation of the primary emulsion influences various properties such as DE stability and encapsulation efficiency. The stability of W1/O and W1/O/W2 emulsions is strongly affected by the composition and physicochemical properties of the oil phase. Although most available vegetable oils usually have relatively high viscosity and higher water solubility than mineral oils, they can form stable multiple emulsions in association with large lipophilic and hydrophilic molecular emulsifiers (Su, Flanagan, Hemar, & Singh, 2006; Su, Flanagan, & Singh, 2008). The oil phase serves as a sort of membrane that separates the two parts of the aqueous phase, influencing the ability of the materials to transport between the inner and the outer phase (Lutz, Aserin, Wicker, & Garti, 2009). The nature of the oil also determines the droplet size, interfacial tension and stability, which in turn may affect the diffusion rates of hydrophilic species retained within the inner aqueous phase through the oil phase (Bonnet et al., 2009; Garti, 1997; Weiss & Muschiolik, 2007). Although kinetic release of compounds entrapped in the inner aqueous phase of the DE depends on the bioactive compound, the release kinetics of compounds such as L-tryptophan (Weiss, Scherze, & Muschiolik, 2005), sodium ascorbate (Lutz et al., 2009) or magnesium (Bonnet et al., 2009) has also been found to be dependent on the type of oil used in the W1/O/W2 emulsion. The objective of this study was therefore to examine the influence of the lipid source used as a lipid phase of the primary emulsion on riboflavin encapsulation and physicochemical properties of food-grade W1/O/W2 emulsions. To that end, various DEs were prepared with different vegetable oils in order to be able to both reduce and improve the FA profile of foods when used as food ingredients. In addition, animal fat was used to fabricate DEs as an alternative strategy for reducing fat content, thus avoiding the use of fat sources that are not habitual in some food matrices.
2.2. Preparation of double emulsions (DEs) A two stage procedure was used to prepare stable DEs, as reported elsewhere (Cofrades et al., 2013) with minor modifications. The inner (W1) phase consisted of a 5.84 g/L aqueous solution of NaCl plus 0.03 g/100 mL riboflavin and was protected from light. The outer (W2) phase was prepared by dispersing 5.84 g/L NaCl plus 0.5 g/100 mL sodium caseinate in distilled water at room temperature until fully dissolved. Thereafter, 0.02 g/100 mL sodium azide was added to the outer phase to prevent microbial growth and allow us to study their stability. The pH of the inner and outer aqueous phases was in the range 5.7e6.5 at all times. The lipid phase (O) consisted of chia, sunflower or olive oil, or RPF (94 g/ 100 g), plus PGPR as the lipophilic surfactant (6 g/100 g). Phases were mixed for 15 min at 60 C in a Thermomix TM-31 food processor (Vorwerk, Germany) at setting 5. Four primary coarse emulsions (W1/O) were prepared by dropwise addition of the inner (W1) aqueous phase (20 g/100 g) to each of the four lipid phases (80 g/100 g) in the Thermomix food processor set at 60 C for 15 min at setting 6. Each primary coarse emulsion was passed twice through a two-stage high pressure homogenizer at 55,000/7,000 kPa (Panda Plus 1000, GEA NiroSoavi, Parma, Italy). The resulting primary fine emulsions (W1/O) were then allowed to cool at room temperature before preparation of the DEs. These were prepared by gradually adding each W1/O fine emulsion (40 g/100 g) to the outer (W2) aqueous phase (60 g/100 g) in the Thermomix food processor set at 37 C and at setting 3. The resulting coarse W1/O/W2 emulsions were passed twice through a two-stage high pressure homogenizer (Panda Plus 1000) at 15,000/3,000 KPa to obtain the final DEs containing chia, sunflower or olive oil or RPF (DEsCO, DEsSO, DEsOO, DEsRPF respectively). These DEs are expected to be used as ingredients for the intended food application immediately or stored for as short a time as possible for a number of reasons (economics, organisation of production tasks, safety, etc.). 2.3. Thermal treatment and storage
2. Material and methods
Immediately after DE preparation, 10 mL aliquots were added to graduated plastic tubes. Half of the tubes were immediately stored at 4 C. The other half were heated for 30 min in a water bath at 70 C and stored at 4 C after cooling. Additionally, heated and unheated DEs prepared with RPF were stored at room temperature (approx. 22 C). All samples were analysed at 1, 3, 6 and 8 days of storage unless otherwise specified.
2.1. Materials
2.4. Physical stability
In ascending order of FA unsaturation, unrefined chia vegetable oil (Primaria Premium Raw Materials, S.L; Valencia, Spain), refined sunflower oil (Koipesol Semillas S.A.; Sevilla, Spain) and tara S.A.; Madrid, virgin olive oil (Carbonell Virgen Extra, SOS Cue Spain) were used in the formation of the lipid phase of W1/O/W2 emulsions because their FA profiles (oils rich in n-3, n-6 and n-9 series respectively) are healthier than those of animal fats (rich in saturated FAs). Approximately 600 g of ground Iberian pork backfat purchased in a local market was heated up to 70 C in a water bath and then filtered through cheesecloth to obtain rendered pork backfat (RPF), for use as the lipid phase in the formation of reduced fat ingredients. Polyglycerol polyricinoleate (PGPR; SUGIN 476/M) was purchased from Cargill S.L.U. (Martorell, Spain). Sodium caseinate was from DMV (Excellion EM 7, DMV Campina B.V.; Veghel, The Netherlands). Sodium chloride was from Panreac (Panreac Química, S.A.; Barcelona, Spain). Riboflavin and sodium azide were from Sigma-Aldrich (Madrid, Spain).
Gravitational separation (creaming) of heated and unheated DEs was recorded in triplicate over storage in terms of phase separation and expressed as percentage of initial sample height. 2.5. Microscopy Light microscopy was used to examine emulsion morphology. Samples were diluted 8 times with 5.84 g/L NaCl, placed on microscope slides and carefully covered with a cover slip. The DE microstructure was observed using a Reichert microscope (Munich, Germany) at 40 and 100 magnifications. Measurements in heated and unheated DEs were made at days 1 and 8 of storage. 2.6. Particle size characteristics The particle size and distribution of oil droplets in the heated and unheated DEs was determined immediately, after 10-fold
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dilution with 5.84 g/L NaCl, with a Malvern Mastersizer S laser diffraction particle size analyser (Malvern Instrument Ltd, Worcestershire, UK) equipped with a He-Ne laser (l ¼ 633 nm). The measurement range was 0.05e900 mm. Obscuration was in the range of 8e15%. Particle size calculations were based on the Mie Scattering theory. Surface average diameter (d32) was measured immediately after addition to the dispersion unit. Results as reported were averages of at least three measurements.
EE ð%Þ ¼ 100
CW2 100 CW1
623
where Cw2 is the concentration of riboflavin determined in the outer aqueous phase, and Cw1 is the concentration of riboflavin considering the amount of the vitamin initially added to the inner aqueous phase and the equivalent dilution produced by mass transfer of water from the inner phase to the aqueous phase. All samples were analysed in triplicate.
2.7. Viscosity 2.10. Statistical analysis The viscosity of the different lipid phases used for the production of DEs and that of the resulting heated and unheated DEs containing vegetable oils was determined at 4 C, whereas the viscosity of RPF and DEsRPF was determined at two temperatures (4 and 25 C). These measurements were done in a CP4/40 coneplate cell (4 angle and 40 mm diameter) with a 150 mm gap on a Bohlin CVO-100 rheometer (Bohlin Instruments Ltd., Gloucestershire, UK) at a constant shear stress of 0.5 s1. Analyses were done at the beginning of storage. Results, expressed as Pa$s, were averages of five determinations. 2.8. Dynamic rheological properties Dynamic rheological experiments were conducted on unheated DEs using a Bohlin CVO-100 controlled-stress rheometer (Bohlin Instruments Ltd., Gloucestershire, UK). The measurement system was a cone plate geometry CP4/40 (4 angle and 40 mm diameter) with a 150 mm gap in all samples. Samples were allowed to relax for 5 min before rheological measurements such as equilibration time after loading the sample on the sensor system. Temperature was controlled with a Peltier Plate system (40 to þ180 C; Bohlin Instruments, Gloucestershire, UK). The linear viscoelastic region was determined for each sample through stress sweeps at 0.1 Hz. After that, a dynamic frequency sweep was conducted by applying a constant pre-determined stress within the linear region, over a frequency range between 0.05 and 1 Hz for each sample. The measurements were made at 25 C. Parameters including elastic modulus (G0 ), viscous modulus (G00 ) and phase angle (d) were derived using the analysis program software. Analyses were done at the beginning of storage. Results were averages of at least three measurements. 2.9. Determination of riboflavin encapsulation efficiency and stability Encapsulation efficiency can be defined as the amount of the bioactive compound which remains entrapped in the inner aqueous phase (W1). The release of riboflavin was determined after each storage period (encapsulation stability) by 4-fold dilution of the DE in a 5.84 g/L aqueous solution of NaCl. Samples were then centrifuged at 2,500 g for 30 min (Heraeus multifuge 3L-R DJB Labcare Ltd.; Buckinghamshire, UK) to separate the fat globules from the outer aqueous phase. These centrifugation conditions did not change d32 after centrifugation. Four mL of the lower aqueous phase was taken out and mixed with 2 mL of 33 g/100 mL trichloroacetic acid. Samples were then filtered through Whatman No. 1 filter paper. The absorbance of the filtrate was measured at 382 nm using a spectrophotometer (Shimadzu UV-1800; Japan). Concentrations were determined from a riboflavin standard curve. The encapsulation efficiency (EE) was calculated with respect to the amount of riboflavin added to the inner aqueous phase using the following equation (Sapei, Naqvi, & Rousseau, 2012):
The effects of the type of lipid phase used (chia, sunflower and olive oils stored at 4 C and RPF stored at room temperature) in the fabrication of the DEs, the thermal treatment (where applicable) and chilling storage of these DEs, on mean particle size, viscosity, physical stability and encapsulation efficiency were analysed using a multifactor ANOVA. Because RPF is solid/semisolid, only the effect of vegetable oils on lipid phase viscosity was included in the statistical analysis. The effects of storage temperature and thermal treatment on the same parameters were considered for DEsRPF stored for 1 day at either 4 C or at room temperature. Interactions between two factors were also considered. A P-value of less than 0.05 was considered significant. Tukey-HSD tests were used to identify statistically significant differences except for viscosity of DEs, for which the Tamhane test was used. Statistical analyses were carried out using IBM SPSS Statistics 21 software. 3. Results and discussion These emulsions must be considered in the context of their practical use. In particular, they are designed for use as ingredients in meat product reformulation processes. Accordingly, all DEs were formulated to contain 2.4 mg of riboflavin per 100 g of DE, since addition to meat systems at levels ranging from 10 to 15 g/100 g is realistic (Cofrades et al., 2013). This means 0.24e0.36 mg of riboflavin per 100 g of food (meat product), which covers 17e25% of the Recommended Dietary Allowances for pregnancy (1.4 mg) (Food Nutrition Board, 1998). As formulated, these DEs were expected to have similar fat contents (32 g/100 g). In meat reformulation processes, the use of RPF in the fabrication of DEs makes it possible to develop novel ingredients to reduce the animal fat content while maintaining sensory properties. Other lipid sources with positive health implications can be used in the preparation of DEs to develop novel ingredients for healthier, low-energy foods. 3.1. Physical stability These DEs have to be prepared beforehand and used as one of a number of ingredients in meat product formulations. Like other perishable meat and non-meat ingredients, they may require cold storage, which needs to be minimized, to prevent microbial growth and maintain stability before they are used as ingredients. These DEs can be heated once they have been added to meat systems to produce cooked meat products, and we therefore also addressed their stability to conventional thermal treatments. All heated and unheated DEs had a homogenous milky white appearance after preparation. As they are for use as fat replacers, this milky appearance is not a problem. Moreover, this appearance was maintained during storage at 4 C for 8 days in those DEs containing vegetable oils. However, heated and unheated DEsRPF samples collapsed and changed to a stiff and creamy homogenous phase after 48 h of cold storage, which limits their utility as food ingredients to that period of time. When DEsRPF was stored at
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room temperature it was found to be stable over time. This suggests that the loss of the initial structure is likely due to the formation of large crystals, causing disruption of the emulsion and concomitant formation of a fat crystal network during storage at 4 C. In an earlier experiment with DEs containing pork lard in the lipid phase and sodium caseinate in the outer water phase, storage at 4 C nezcaused the formation of some clumps (Bou, Cofrades, & Jime Colmenero, 2014). Therefore, it seems that chilling storage of DEs containing fats causes some instability problems, probably due to the high melting point of the lipid phases. The fact that the initial structure of DEsRPFs was maintained within the first 48 h of storage at 4 C is enough to warrant their use in the meat industry. Nevertheless, we decided to compare DEsRPF stored at room temperature with DEs containing vegetable oils stored at 4 C. At the starting time, samples DEsSO and DEsOO showed no signs of creaming (Table 1). In contrast, DEsCO and DEsRPF (this last stored at room temperature) showed little phase separation. At the end of the storage period, phase separation was higher (21.3%) in DEsRPF stored at room temperature, while creaming varied in DE containing vegetable oils (Table 1). Overall, the physical stability of the DEs containing vegetable oils was acceptable and in good agreement with other very similar DEs containing olive oils (Cofrades et al., 2013). Of the DEs containing vegetable oils, the phase separation was 14.0% in DEsOO, decreasing with increasing unsaturation in the lipid phase. Creaming was most intense in DEsRPF, but these samples were stored at room temperature. However, their stability was not very different from that of DEs containing pork lard and stored at 4 C. Bou et al. (2014) compared the stability of DEs containing olive oil and pork lard and reported that, under storage at 4 C, DEs containing olive oil were less stable than DEs containing pork lard (89% and 97%, for olive oil and pork lard respectively). Although temperature is critical for DE stability, it is reasonable to assume that DEsRPFs are quite stable to creaming. Only DEsCO and DEsRPF samples were affected by thermal treatment (data not shown). When DEsCO and DEsRPF (this last stored at room temperature) were heated, phase separation was greater than in the unheated DEs (5.7 vs 7.4% and 11.9 vs 12.9% for unheated versus heated DEsCO and DEsRPF respectively). 3.2. Morphology Fig. 1 shows the microscopic structure of unheated DEsOO, which is representative of the rest. It can be observed that a large
Fig. 1. Light microscopy images of unheated W1/O/W2 emulsions containing olive oil after 8 days of storage at 4 C. Scale bars represent 10 mm at 40 and 100 magnifications.
amount of small oil globules coexist with only some scarce bigger droplets. Irrespective of the size, all droplets have the characteristic structure of a DE, consisting in small water droplets located inside an oil globule; however, because they are small, this structure can be more easily observed in these larger droplets at 100 magnification (Fig. 1 insert). It is important to note that this compartmentalized structure was also observed in all DEsRPFs. However, the structure collapsed in the course of storage at 4 C (not shown). In an earlier experiment, it was also observed that the visual appearance and overall microscopic structure of DEs containing olive oil did not differ greatly from those containing pork lard (Bou et al., 2014). The different lipid sources used to fabricate DEs therefore seemed to have very little effect on their morphology. DEsRPF was unstable after 8 days of storage at 4 C and, as noted earlier, the appearance changed from milky to creamy. However, DEsRPF stored at room temperature and DEsCO, DEsSO and DEsOO stored at 4 C were apparently unchanged after 8 days of storage (not shown). The DEs were heated at 70 C for 30 min to mimic a conventional thermal treatment in the food industry (e.g. meat processing) and thereafter stored under the same conditions as unheated DEs. The compartmentalized structure was unaffected by heating and there
Table 1 Particle size (d32), physical stability (creaming) and encapsulation stability of W1/O/W2 emulsions as affected by the lipid phase.a Day
DEsCO
DEsSO
DEsOO
DEsRPF
Stored at 4 C Particle size (d32; mm)
Physical stability (%)
Encapsulation stability (%)
1 3 6 8 1 3 6 8 1 3 6 8
1.58aX 1.24aX 2.02bX 2.10b 0.7aY 2.8abY 5.2abX 6.8bV 85.4dZ 78.6bX 80.9cY 76.6aX
± ± ± ± ± ± ± ± ± ± ± ±
0.27 0.38 0.10 0.11 0.51 2.86 3.86 3.87 0.79 0.25 0.94 1.06
Stored at 22 C 2.32abY ± 2.49bZ ± 2.28aZ ± 2.20a ± 0aX 2.1bX ± 4.8cX ± 11.2dX ± 81.6bXY ± 82.5bY ± 81.4abY ± 80.4aY ±
0.06 0.13 0.19 0.09 0.20 0.41 0.75 0.60 0.40 0.72 1.46
2.20Y ± 2.16Y ± 2.22YZ ± 2.10 ± 0aX 1.3bV ± 6.8cY ± 14.0dY ± 80.4bX ± 75.4aV ± 75.2aX ± 76.7aX ±
0.17 0.29 0.10 0.19 0.41 0.75 0.89 0.55 0.75 1.37 1.63
2.18Y 2.07Y 2.05XY 2.06 1.12aZ 8.0bZ 19.2cZ 21.3dZ 82.1aY 85.3cZ 84.3bcZ 83.2abZ
± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.10 0.05 0.11 0.20 0.63 0.75 1.21 1.94 0.64 0.79 0.72
Means within the same column without a common letter (aed) are significantly different (P 0.05). Means within the same row without a common letter (VeZ) are significantly different (P 0.05). a DEsCO, DEsSO, DEsOO and DEsRPF stand for double emulsions containing chia oil, sunflower oil, olive oil and rendered pork backfat respectively. Results for DEsRPF stored at 4 C are not shown because this emulsion was not stable throughout storage. Values given in this table correspond to least-square means ± standard deviation obtained from MANOVA (n ¼ 48).
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were no appreciable changes in their morphology (results not shown). Therefore, all these DEs can be used in the development of foods that require some thermal treatment and hence may be suitable for use as low fat ingredients in the processing of many food products. 3.3. Particle size characteristics Regardless of the lipid source and storage time, the DEs displayed unimodal distributions, although with differences in width (Figs. 2 and 3). Unimodal distribution has also been reported in other similar DEs in which PGPR and sodium caseinate were used respectively as lipophilic and hydrophilic emulsifiers (Cofrades et al., 2013; Frasch-Melnik, Spyropoulos, & Norton, 2010). At the start, d32 of DEsSO, DEsOO and DEsRPF were similar, while the droplet diameter of the DE containing the most unsaturated lipid source (DEsCO) was smaller (Table 1 and Fig. 2). These sizes are in agreement with other very similar DEs containing olive oil and pork lard (Bou et al., 2014). Bonnet et al. (2009) compared different lipids (olive oil, rapeseed oil, olein and miglyol) for use in the formulation of DEs. These authors reported similar droplet diameters regardless of the lipid source used. Conversely, other authors found different sizes when examining the effect of different semicrystalline lipids on the average lipid droplet diameter of the resulting DE, although they reported no clear trend and offered no explanation (Weiss et al., 2005). The comparison between DEsRPF stored for 1 day at 4 C and stored at room temperature showed that droplets were larger in the latter case (2.18 vs 2.00 mm for DEs stored at room temperature and at 4 C respectively). Thus, the nature of the lipid used in DEs can influence lipid globule sizes to some extent, but other factors such as viscosity, W1 droplet size and interfacial tension have also been reported to influence particle size and need to be considered to understand and prepare DEs with tailored properties (Weiss & Muschiolik, 2007). DEsSO, DEsOO and DEsRPF (the latter stored at room temperature) showed no increase in oil globule sizes after 8 days of storage (Table 1). In the case of DEsCO, the oil droplet size was unchanged up to 3 days of storage at 4 C but increased over longer storage times (Table 1, Fig. 3). This increase in DEsCO was a factor in the absence of differences between DEs with different lipid sources at the end of the storage period. Chia oil is very rich in linolenic acid (Ayerza & Coates, 2009), and therefore it displayed lower viscosity
Fig. 3. Particle size distribution of W1/O/W2 emulsions containing chia oil after 1 day and 8 days of storage at 4 C.
(0.148 Pa s) because it was more unsaturated (Table 2), which in turn may have contributed to this difference in behaviour. The thermal treatment applied (30 min at 70 C) had no effect on oil droplet distribution or average droplet diameter (global means were 2.06 and 2.09 mm for fresh and heated DEs respectively). And again, the thermal treatment had no effect when DEsRPF samples stored at two different temperatures were compared. Bonnet et al. (2009) studied stability of DEs containing olive oil to thermal treatments by heating them for 20 s at 72 C and 20 s or 5 min at 92 C. As in the present case, the authors reported that the DEs retained the characteristic DE structure and lipid droplet sizes were unaffected. 3.4. Viscosity The influence of the type of lipid on the viscosity of the different lipid phases (containing 6 g/100 g PGPR) used for the formation of primary emulsions is shown in Table 2. As expected, viscosity decreased with increasing unsaturation of the lipid phase. The stability to creaming of DEs stored for 8 days at 4 C seems to be in inverse relation to lipid phase viscosity (Tables 1 and 2). The physical stability of DEs depends on their viscosity because this hinders phase separation. All DEs showed low viscosities, possibly because this is mainly determined by the viscosity of the
Table 2 Viscosity of lipid phases and their respective W1/O/W2 emulsions.a
Fig. 2. Particle size distribution of W1/O/W2 emulsions containing chia oil (DEsCO), sunflower oil (DEsSO), olive oil (DEsOO) and rendered pork backfat (DEsRPF) after 1 day of storage at 4 C.
Lipid used
Temperature at measurement
Lipid phase (Pa$s)
Unheated W1/O/W2 (kPa$s)
Heated W1/O/W2 (kPa$s)
Chia oil Sunflower oil Olive oil RPF RPF
4 C 4 C
0.148 ± 0.0034a 0.174 ± 0.0051b
31.6 ± 0.4cX 24.0 ± 0.7aX
32.8 ± 0.1cY 24.2 ± 0.9aX
4 C 4 C 25 C
0.253 ± 0.0016c 380 ± 9.8 3.6 ± 0.54
26.8 ± 1.4bX 273 ± 68dX 139 ± 36X
26.8 ± 0.1bX 209 ± 79dX 187 ± 24X
Means measured at 4 C and within the same column without a common letter (aed) are significantly different (p 0.05). Means for W1/O/W2 emulsions within the same row without a common letter (XeY) are significantly different (p 0.05). a The lipid phase contains 6% polyglycerol polyricinoleate. Values are means ± standard deviation of lipid phases and W1/O/W2 emulsions measured at the beginning of storage. All emulsions were stored 1 day at 4 C except those containing rendered pork backfat (RPF) which were stored at room temperature (approx. 22 C).
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continuous phase, which was very low (Table 2). The external aqueous phase was the same for all DEs, and so the differences in viscosity could be directly attributed to the different lipid phases. In fact, the viscosities of DEsRPF were higher than those of DEs containing vegetable oils (Table 2). In the case of DEs containing vegetable oils, the viscosities of DEsOO and DEsSO were similar and lower than the rest. Therefore, the low viscosities found in DEsSO and DEsOO could explain the low stability of the DEs to creaming. However, DEsCO, which was the most unsaturated, showed the highest viscosity of all the vegetable oils. There are various possible explanations for these results. On the one hand, the viscosity of DEs has been associated with the particle size and particle size distribution and hence is a factor determining their stability. The fact that the lipid droplets were initially smaller could explain why DEsCO was the most stable to creaming (Tables 1 and 2). Moreover, the narrower the particle size distribution, the greater was the DE's viscosity and stability to creaming. Such a narrow distribution increases the probability of particleeparticle interactions and would cause the DEsCO to be more resistant to gravity, so that the system is more stable to creaming (Fig. 2). On the other hand, those emulsions with a wide droplet size distribution, as in the case of DEsSO (Fig. 2), had more free space for particles to move around, making it easier for the sample to flow, meaning that it was less viscous (Table 2). Alternatively, the higher viscosity of DEsCO could also be explained by the presence of minor compounds such as mucilages (soluble fibre) that can be present in this type of non-refined oil. These mucilages can bind the water of the DE and could thus explain the observed increase in the viscosity of DEsCO. Moreover, the presence and hydration of mucilages as a consequence of the thermal treatment applied may explain why the viscosity of DEsCO increased while the viscosities of the other DE remained unaffected (Table 2). 3.5. Dynamic rheological properties Dynamic oscillatory shear tests were used to characterize the viscoelastic properties of the DEs containing different types of fat (vegetable or animal). The results from the oscillatory shear tests were expressed in terms of the variation of elastic modulus (G0 ), viscous modulus (G00 ) and phase angle (d) as a function of frequency (Hz). The dynamic mechanical spectra of all DEs showed that the viscous modulus (G00 ) values were higher than the corresponding storage modulus (G0 ) values over the entire frequency range, with G0 values too low and irregular for a Power Law to fit. Similar results were found in DEs formulated with olive oil and pork lard, nonpressurized and pressurized at 400 and 600 MPa (Bou et al., 2014). This observation suggests that viscous behaviour predominates over elastic behaviour; all DEs exhibited liquid-like behaviour regardless of the type of oil used. In all samples, G00 increased and the phase angle remained roughly constant when the frequency increased (Fig. 4a and b). Since the phase angle indicates the viscoelasticity index of a sample, this can be interpreted in terms of “solid-like” emulsions (d ~ 0 ) and “liquid-like” emulsions (d ~ 90 ); the predominantly viscous nature of the emulsions was confirmed by the liquid-like aspect and d values of the DE, which ranged from 78 to 85 (Table 3). Ross-Murphy (1995) described changes in and dependency of G00 and G0 of biopolymers with frequency, indicating that a dilute solution typically has a high G00 to G0 ratio, with both moduli showing a strong frequency dependence. When a system shifts from a fluid to a more solid-like system its G0 increases and crosses over G00 . Such a system is described rheologically as a concentrated solution. However, in our systems no changes in the behaviour of G00 and G0 were observed anywhere within the frequency range. Thus, our DEs retained the dilute
Fig. 4. Variation of the viscous modulus (G00 ) and phase angle (d) as a function of frequency (Hz) as determined in the frequency sweep tests (at 25 C) in W1/O/W2 emulsions. A) G00 (Pa) for unheated W1/O/W2 emulsions made with different fat sources; B) d ( ) for unheated W1/O/W2 emulsions made with different fat sources. See Table 1 for abbreviations.
solution structure, without any major conformational changes over the frequency range studied. This behaviour could be related to the high water content in the double emulsions (~70 g/100 g). Similar results were observed by (Lee, Anema, & Klostermeyer, 2004) in processed cheese spread samples with a water content above 54 g/ 100 g, and they found that G00 was greater than G0 over the entire frequency range studied, with no cross-over at low frequencies. These authors reported that small increases in water content resulted in roughly twofold decreases in G0 and G00 ; however, the phase angle increased with increasing moisture. As there was no change in the water content of our samples, there were no changes Table 3 Fitted values for the viscous modulus as determined in the frequency sweep test for unheated W1/O/W2 emulsions formulated with different lipid sources.a Double emulsion
G00 0 (Pa)
DEsCO DEsSO DEsOO DEsRPF
0.066 0.052 0.059 0.087
± ± ± ±
n00 0.001 0.001 0.001 0.001
0.801 1.077 1.022 0.354
d ( ) ± ± ± ±
0.01 0.01 0.01 0.01
85.03 81.55 84.27 83.66
± ± ± ±
3.37 2.44 3.70 3.37
a DEsCO, DEsSO, DEsOO and DEsRPF stand for double emulsions containing chia oil, sunflower oil, olive oil and rendered pork backfat respectively.
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in the phase angle (Fig. 4). However, according to Brummer (2006), the mechanical spectrum of a stable emulsion with greater internal strength has a storage modulus, G0 , higher than its viscous modulus, G00 , and both moduli should be almost parallel throughout the observed frequencies, with a slight increase in the slope at high frequencies. In this respect, DEsRPF behaved more like a stable emulsion since it showed a smaller increase with the frequency (
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contain a relatively large amount of free FAs (acid value 1 g/ 100 g), and these have been reported to migrate and concentrate in the oilewater interfaces, thus changing the properties and affecting the oxidative stability of oil-in-water emulsions (Waraho, Cardenia, Rodriguez-Estrada, McClements, & Decker, 2009). Also, monoacylglycerides and diacylglycerides that are normally present in olive oils have been shown to be good surfactants. It is therefore reasonable to suppose that these minor compounds may play a role in internal interface stability and in turn may affect the encapsulation of bioactive compounds, probably facilitating the transport of entrapped compounds from the inner water phase. The high viscosity of DEsRPF (Table 2) could explain why the initial riboflavin encapsulation efficiency of DEsRPF was unaltered after 8 days of storage at room temperature (Table 1). DEsRPF thus displayed good riboflavin encapsulation stability (83.2%) at the end of the storage period (Table 1). These results are in line with the report by Weiss et al. (2005) that the release of encapsulated tryptophan was reduced by increasing the solid fat content in the oil phase. These findings can be explained by the fact that semicrystalline lipid phases promote not only increased viscosity (Table 2) but also effective encapsulation. On the other hand, storing these DEs at temperatures below or close to the lipid phase melting point can be critical for riboflavin encapsulation. Finally, it is important to note that the thermal treatment had no effect on riboflavin encapsulation (overall least squares means were 80.6% and 80.7% for unheated and heated DEs respectively). Therefore, bioactive compounds can be encapsulated in the inner water phase, but they can also be entrapped with this technology following a thermal treatment. Thus, DEs can be used as ingredients to develop novel functional food products, such as meat products, that require some kind of thermal treatment. 4. Conclusions DEs can be used to develop healthier and functional foods that can be subjected to thermal treatments while still entrapping bioactive compounds such as riboflavin. In this experiment riboflavin was efficiently encapsulated, although this also depended on the nature of the lipid phase used. Of the lipid phases used to prepare DEs, the ones containing chia oil were the most efficient in encapsulating riboflavin. However, during storage at 4 C, the chia oil DE released riboflavin more rapidly than the ones containing sunflower or olive oil. At this storage temperature, DEs containing RPF need to be used within 48 h because of DE breakdown. Nonetheless, these DEs were stable when stored at room temperature for 8 days and under these conditions exhibited the greatest (83.2%) riboflavin encapsulation stability. Acknowledgements R. Bou has been supported by a contract from the JAE-postdoctoral (CSIC) Program. This research has been supported by project AGL 2011-29644-C02-01 from the Spanish Ministry of Science and Innovation. Primaria Premium Raw Materials, S.L. kindly provided the chia oil. The authors wish to thank A. del Hoyo for her help during analyses. References Ayerza, R., & Coates, W. (2009). Influence of environment on growing period and yield, protein, oil and a-linolenic content of three chia (Salvia hispanica L.) selections. Industrial Crops and Products, 30, 321e324. de Benoist, B., McLean, E., Egli, I., & Cogswell, M. (2008). Worldwide prevalence of anaemia 1993e2005. WHO global database on anaemia WHO Library Cataloguing-in-Publication Data http://whqlibdoc.who.int/publications/2008/ 9789241596657_eng.pdf.
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