W emulsions stabilized by PGPR and lecithin

Food Research International 122 (2019) 252–262

Contents lists available at ScienceDirect

Food Research International journal homepage: www.elsevier.com/locate/foodres

Formation and stability of W/O-high internal phase emulsions (HIPEs) and derived O/W emulsions stabilized by PGPR and lecithin Paula K. Okuro1, Andresa Gomes1, Ana Letícia R. Costa, Matheus A. Adame, Rosiane L. Cunha

T

⁎

Department of Food Engineering (DEA), Faculty of Food Engineering (FEA), University of Campinas (UNICAMP), Rua Monteiro Lobato, 80; Campinas-SP; CEP, 13083862, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Soybean lecithin Food emulsion Texture modification Fat replacer Phases inversion Sunflower oil Natural emulsifier

Water-in-oil high internal phase emulsions (HIPEs) can provide interesting textures that could be used to reduce trans- and/or saturated fat content in food products. On the other hand oil-in-water emulsions can be found in a variety of food and beverages. Moreover, strategies aiming synthetic or semi-synthetic ingredients replacement by natural alternatives for food applications has been pursuit. For these purposes, the effect of partial replacement of PGPR by lecithin on properties of either W/O-HIPEs or O/W emulsions manufactured from the same initial composition but showing different volume fraction of dispersed phase were investigated aiming to understand the behaviour of emulsifiers' mixture in water-oil or oil-water interfaces. Firstly, water-in-oil HIPEs were produced using a rotor-stator device. At fixed total amount of emulsifier (2% w/w), W/O emulsions stabilized with LEC:PGPR ratios of 0.5:1.5 and 1.0:1.0 showed similar droplet size with a better kinetic stability compared to emulsions containing only PGPR. These results indicated good interaction between LEC and PGPR, which was also confirmed by dynamic interfacial tension profile and interfacial dilational rheology. In order to reduce the droplet size of W/O-HIPEs, these emulsions were subsequently subjected to high-pressure homogenization and interestingly phases inversion was observed. Confocal microscopy confirmed the phases inversion attributed to high input of energy leading to the formation of O/W emulsions. Then both W/O-HIPEs and O/W emulsions were investigated regarding LEC:PGPR mixtures as emulsifiers. All W/O-HIPEs showed shear thinning behavior and high viscosity at low shear rate whereas O/W emulsions showed low viscosity and Newtonian behavior. The increase of lecithin content in emulsifier mixture led to more stable O/W emulsions, whereas more stable W/O-HIPEs were produced by lecithin and PGPR mixtures ratio of 0.5:1.5 and 1.0:1.0.

1. Introduction

extensively studied with a number of emulsifiers envisioning provide kinetic stability through steric or electrostatic mechanisms that inhibit oil droplet aggregation (Jafari, Assadpoor, He, & Bhandari, 2008). The stability of both W/O and O/W emulsions are influenced by the aqueous or oil phase properties, by the presence of other compounds entrapped in the system as well as by type and concentration of emulsifier. Polyglycerol polyricinoleate (PGPR) is a hydrophobic semi-synthetic emulsifier widely used to stabilize W/O emulsions such as margarines, butter, salad dressing and chocolate (Márquez et al., 2010). This emulsifier is obtained from esterification reaction of polymerized glycerol with condensed castor oil fatty acids (Dedinaite & Campbell, 2000; Gülseren & Corredig, 2014). PGPR is considered GRAS (Generally Recognized As Safe) by FDA, although its use in food products has to be indicated on labels and a maximum dosage about 2.6 mg.kg−1 of body weight per day is recommended (Alicja et al., 2017; Wilson & Smith,

Water-in-oil (W/O) emulsion is a class of emulsion less reported than its analogue oil-in-water (O/W) emulsion, although it shows interesting functional applications (Márquez, Medrano, Panizzolo, & Wagner, 2010). Most of traditional W/O food emulsions are stable solid or semi-solid systems such as margarine and butter which are stabilized by fat crystals (Prichapan, McClements, & Klinkesorn, 2017). On the other hand, fluid/liquid W/O emulsions show a high droplet mobility making them susceptible to destabilization mechanisms as sedimentation, flocculation and coalescence (McClements, 2015; Ushikubo & Cunha, 2014). Oil-in-water (O/W) emulsions are present as integral part of many food products such as milk, dressings, desserts, and beverages (Bai, Huan, Li, & McClements, 2017; Dickinson, 2015). This system has been

⁎

Corresponding author. E-mail address: [email protected] (R.L. Cunha). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.foodres.2019.04.028 Received 24 May 2018; Received in revised form 10 April 2019; Accepted 11 April 2019 Available online 13 April 2019 0963-9969/ © 2019 Published by Elsevier Ltd.

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

the effect of partial replacement of PGPR by lecithin on properties of either W/O-HIPEs or O/W emulsions manufactured from the same initial composition but different volume fraction of dispersed phase aiming to understand the behaviour of emulsifiers' mixture in distinct water-oil or oil-water interfaces.

1998). Consumers are increasingly demanding healthier products that have been encouraging the total or partial replacement of synthetics or semi-synthetics ingredients in food products formulations by natural alternatives (Belayneh, Wehling, Cahoon, & Ciftci, 2018; Gülseren & Corredig, 2014). Therefore new approaches to reduce the usage of PGPR are highly sought after (Gülseren & Corredig, 2012). Conversely, lecithins are amphiphilic molecules extracted from natural sources as soybean, wheat, oat or eggs, and they are important components of cell membranes (Kralova, Sjöblom, Kralova, & Sjöblom, 2009). The behavior of lecithin as surface-active compound is not totally understood because of its complex structure and variable internal composition consisting of phospholipids and sphingolipids (Belayneh et al., 2018). Although lecithin can be applied as emulsifier in a wide variety of emulsions, this ingredient has limited functionality concerning its multicomponent nature (Dashiell, 1989). Thus, surface activity and performance of commercial lecithin depends on its composition since some phospholipids are more effective in stabilizing of water-in-oil and others in oil-in-water emulsions (Joshi et al., 2006). Phospholipids can be modified by distinct treatments (deoiled, enzyme modified, choline-enriched) resulting in products with different degree of purity and specificity (Guiotto, Cabezas, Diehl, & Tomás, 2013; Kralova et al., 2009). However the purification process increases the cost of the product in around five times in relation to those non-purified soybean lecithins (Yokota, Moraes, & Pinho, 2012). In response to this demand, some strategies have been proposed to decrease the use of synthetic or semi-synthetic ingredients. For instance, the addition of a second hydrophobic emulsifier or addition of a hydrophilic surfactant to water phase have been studied (Gülseren & Corredig, 2014). In this vein, the right choice of the emulsifiers and the appropriate ratio in the mixture are necessary to provide technological functionality to the hybrid system. Health related problems associated with the diet quality such as obesity, hypertension and cardiovascular diseases have been considered as a big issue. The need of fat intake reduction and the consumers demand for healthier products have encouraged efforts from both academic and industrial researches. However, it is known that fat plays a key role in texture properties of food products as plasticity, mouthfeel, hardness, crispness, spreadability, consistency among others. In this sense, High Internal Phase Emulsion (HIPE) appears as alternative for (semi-)solid fats. HIPEs are characterized by their high disperse phase volume ratio (ϕ) of 0.74 or greater, showing droplets tightly packed and the external or continuous phase acting as a liquid film giving to these emulsions a highly viscous characteristic (Yang et al., 2018; Zhang et al., 2016). Furthermore the consumption of rich-creaminess food (usually with high viscosity) is related to the increase of expectations of satiation and satiety regardless the energy content of products (Lett, Norton, & Yeomans, 2016). Thus, HIPEs emerge as an interesting approach to increase viscosity of liquid W/O emulsions, decreasing their kinetic instability. Besides texture modifier, HIPEs have been applied as clouding agent in cosmetic and pharmaceutical formulations (Zeng et al., 2017). This research evaluated the partial replacement of PGPR by natural lecithin aiming to decrease semi-synthetic emulsifier amount in W/O emulsions showing a high dispersed phase. This system can be considered promising since even with high amount of water (dispersed phase) it is still able to impart structure and act as texture modifier. It was hypothesized that PGPR would interact with the hydrophobic moieties of lecithin causing changes in the viscoelastic properties of the interface. Although it is known that the presence of PGPR dominates the elastic properties of the interfacial film, even at fairly low concentrations (Gülseren & Corredig, 2012, 2014). Furthermore, the process effect and the replacement of PGPR by a cheaper and natural ingredient were considered based on the effect of high pressure homogenization and use of commercial non-purified soybean lecithin, respectively. In view of that, the objective of the present work was to investigate

2. Material and methods 2.1. Material Polyglycerol polyricinoleate (GRINDSTED® PGPR) was kindly donated by Danisco Brasil Ltda (Brazil) and sunflower oil (Bunge Alimentos S.A., Brazil) was purchased in the local market. The enzymemodified fluid lecithin Solec™ AEIP was donated by The Solae Company™ (St. Louis, USA). This lecithin contained 16% (w/w) phosphatidylcholine (PC), 15% (w/w) lysophosphatidylcholine (lyso-PC), 14% (w/w) phosphatidylethanolamine (PE), 12% (w/w) glycolipids and minor phospholipids, 6% (w/w) of moisture and sugars and 37% (w/w) triglycerides. 2.2. Emulsion preparation 2.2.1. Water-in-oil high internal phase emulsions High internal phase water-in-oil (W/O) emulsions were prepared using a fixed aqueous to oil phases ratio (75:25). The oil phase comprised of sunflower oil and emulsifier mixture with different LEC:PGPR ratios (0:2.0, 0.5:1.5, 1.0:1.0 and 1.5:0.5% w/w) and water was used as aqueous phase. Initially the emulsifier mixture was dissolved in sunflower oil for 15 min using a magnetic stirrer at 42 ± 2 °C. Then this dispersion was added to a jacketed vessel attached to a thermostatic bath (Quimis, Brazil) at 25 ± 2 °C. Afterwards the aqueous phase was added dropwise to the oil dispersion (sunflower oil + emulsifier mixture) using a peristaltic pump Masterflex L/S (Cole-Parmer Instrument Company, EUA) with a mean flow rate of 2.5 ml/min. At the same time this mixture was homogenized with a rotor-stator system (Ultra Turrax T18, IKA, Germany) at 14,000 rpm. When the aqueous phase was completely incorporated into the emulsion (after 50 min), the rotational speed was decreased to 11,000 rpm and the emulsion was homogenized for additional 4 min. During production of W/O-HIPEs stabilized only by lecithin (2% w/w) a drastic drop in viscosity was observed before to complete the full incorporation of water indicating phases inversion from W/O to O/W. Therefore, this condition was not evaluated. Apart from this condition (2% w/w lecithin), all other mixtures comprised of different LEC:PGPR ratios (1.5:0.5; 1.0:1.0; 0.5:1.5) and PGPR were evaluated. 2.2.2. Oil-in-water emulsions Oil-in-water emulsions were further obtained by submitting W/OHIPEs (previously manufactured as described above) to high pressure homogenization at 20 MPa/5 MPa using a Panda 2 K NS1001L doublestage homogenizer (Niro Soavi, Italy). This strategy was carried out in an attempt to reduce even more the droplet size of W/O-HIPEs. However, it caused the phases inversion producing O/W emulsions and we considered interesting to compare this emulsion with HIPE W/O emulsions since they have the same composition but different dispersed and continuous phases. Therefore both water-in-oil and oil-in-water emulsions were prepared in two replicates and they were evaluated from particle size distribution, optical and confocal microscopy, rheological behavior and kinetic stability (backscattering). In addition, characterization of the oil and aqueous phase was investigated by means of dynamic interfacial tension and interfacial dilational rheology. 2.3. Particle size distribution The droplet size distribution was determined by the laser diffraction 253

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

2.6. Kinetic stability - laser scanning turbidimetry

method using a Mastersizer 2000 (Malvern Instruments Ltd., Malvern, UK). Emulsions were dispersed in their corresponding continuous phase: sunflower oil for W/O-HIPEs and water for O/W emulsion (refractive indexes 1.4694 and 1.33, respectively). Ultrasound was applied for 2 min before the measurements to avoid the presence of bubbles. The rotational velocity was 2300 rpm for all emulsions. Volume based particle distribution was presented to illustrated the presence of big and small droplets and eventual coalescence events. The droplet size was expressed as the volume-surface mean diameter (D32) calculated according to Eq. (1). Measurements were performed just after emulsion preparation.

∑ ni di3 ∑ ni di2

D32 =

Emulsion stability was monitored with the optical scanning instrument Turbiscan ASG (Formulaction, France). Freshly prepared emulsions were placed in flat-bottomed cylindrical glass tubes (140 mm height, 16 mm diameter) and the first measurement of backscattered light intensity was performed (0 day). The tubes were stored at 25 °C during 7 days before to perform the second measurement of backscattered light intensity obtained at wavelength of 880 nm. Emulsion destabilization was analyzed using backscattering (BS) profiles at different sample height (mm). A sample height of 0 mm corresponds to the bottom of the measurement cell. Simultaneously Turbiscan Stability Index (TSI) was measured as an indication of destabilization process of the emulsions. This index is calculated as the sum of all the destabilization processes in the measuring cell according to Eq. (4).

(1)

where ni is the droplets number with diameter di.

TSI =

2.4. Microstructure

∑ |scanref (hj) − scani (hi)| j

(4)

2.4.1. Optical microscopy The emulsion microstructure was observed in an optical microscope (Axio Scope.A1, Carl Zeiss, Germany) with 100× oil immersion objective lens. The images were captured with the software AxioVision Rel. 4.8 (Carl Zeiss, Germany). The optical microscopy was performed on the fresh emulsions and after seven days of storage.

wherescanrefandscaniare the initial backscattering value and the backscattering value after 7 days of storage, respectively, hjis the given height in the measuring cell and TSI is the sum of all the scan differences from the bottom to the top of the vial.

2.4.2. Confocal scanning laser microscopy (CSLM) Sunflower oil used to form the emulsions was stained with fluorescent dye Red Nile. Fresh W/O and O/W emulsions were examined using a Zeiss LSM 780-NLO confocal on an Axio Observer Z.1 microscope (Carl Zeiss, Germany) with an objective 100×. The images were collected using 530 nm laser lines for excitation of the Red Nile, with pinholes set to 1 airy unit for each channel and 1024 × 1024 image format.

The following analyzes were performed with the aim to evaluate the influence of the PGPR-lecithin (LEC) mixtures on the interfacial properties between oil and water.

2.7. Characterization of the oil and aqueous phase

2.7.1. Dynamic interfacial tension The interfacial tension was measured at 25 °C using a tensiometer Tracker-S (Teclis, France) by the pendant droplet method. Initially lecithin LEC:PGPR mixtures were dispersed in sunflower oil. The droplet volume were 5.0, 4.0, 3.0, 1.5 and 2.0 mm3 to LEC:PGPR ratio of 0:2.0, 0.5:1.5, 1.0:1.0, 1.5:0.5 and 2.0:0, respectively. These dispersions were heated at 42 ± 2 °C for 15 min under magnetic stirring at 400 rpm, with subsequent cooling at room temperature. Then dispersions were diluted in sunflower oil (1:4) since the non-diluted dispersion showed high opacity hindering image analysis system to assess the dynamic surface tension. Measurements were performed in triplicate.

2.5. Rheological measurements Rheological measurements of W/O-HIPEs were carried out using a Physica MCR301 modular compact rheometer (Anton Paar, Austria) with stainless steel cone-plate geometry (diameter = 49.968 mm; gap = 0.208 mm). Flow curves of O/W emulsions were obtained using a stress-controlled rheometer (AR1500ex, TA Instruments, England) with double concentric cylinders geometry consisting of an inner cylinder (outer radius = 17.53 mm, inner radius = 16.02 mm) and an outside cup (outer radius = 18.45 mm, inner radius = 15.10 mm). The gap for double concentric cylinders was fixed in 0.5 mm. The shear rate varied between 0 and 300 s−1 and the flow curves were obtained in sequential three flow steps: up-down-up cycles. The third flow curve data were fitted to the models for power-law (Eq. (2)) and Newtonian fluids (Eq. (3)). Emulsions were evaluated approximately four hours after their preparation at 25 °C.

2.7.2. Interfacial dilational rheology Dilational dynamic experiments were performed using a tensiometer Tracker-S (Teclis, France) at 25 °C subjecting the droplet interface to very low sinusoidal compression and expansion of the surface area Α. The measurement of the interfacial dilational modulus E (Eq. (5)) is a result of a dilational change of interfacial tension γ (dilational stress) (Eq. (6)) as a consequence of a small change in interfacial areaΑ (dilational strain) (Eq. (7)) (Lucassen & Van Den Tempel, 1972).

E=

•

σ = k (γ )n

(2)

•

σ = ηγ

(3) •

where σ is the shear stress (Pa), η is the viscosity (Pa·s), γ is the shear rate (s− 1), k is the consistency index (Pa·sn) and nis the flow behavior index (dimensionless). Curve fitting was carried out using the Solver function in Microsoft Excel to determine the rheological parameters, kand n in the Power law model (Eq. (2)). To define the best fit a minimum sum of square errors (SSE) was used as the criteria during fitting. The goodness of fit, R2 was calculated as R2 = 1 - (SSE/SST), where SST is the total corrected sum of squares. For non-Newtonian fluids, the apparent viscosity, was reported as a function of shear rate.

dγ = E′ + iE " d ln A

(5)

γ = γ0 + ∆γ sin(ωt + δ )

(6)

A = A0 + ∆A sin(ωt )

(7)

where ω is the angular frequency (Hz), the parameters γ0 and A0 are the equilibrium reference of the interfacial tension (N/m) and the unperturbed interfacial area (m2) of the drop, respectively; Δγ and ΔA are the measured change in stress and strain amplitude respectively, and δ is the phase angle between stress and strain. The linear viscoelastic region was previously determined by amplitude sweep tests, with deformation varying between 5 and 10% at a fixed frequency of 0.1 Hz. A deformation of 6% at a frequency of 0.1 Hz was chosen to perform the measurements of dilational rheology. Firstly, 254

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

8

Table 1 Mean droplet diameter (D32) of W/O-HIPEs and O/W fresh emulsions.

0: 2.0 0.5: 1.5 1.0: 1.0 1.5: 0.5

W/O-HIPEs

O/W emulsion

D32 (μm)

D32 (μm)

2.1 2.4 2.4 4.0

± ± ± ±

0.2C 0.3 BC 0.1 B 0.3 A

4.9 3.2 2.5 1.9

± ± ± ±

6 Volume (%)

Emulsifier (% w/w) (Lecithin: PGPR)

A)

0.3 A 0.0 B 0.1C 0.0 D

4

2 Different letters indicate significant difference at p < .05 in the same column.

0

a droplet of oil was generated and the interfacial tension was measured for 5 min. After that, five sinusoidal oscillation cycles were performed followed by a time corresponding to 30 cycles without any oscillation.

0.01

0.1

1

10

100

1000

0.1

1

10

100

1000

8

B) 2.8. Statistical analysis 6 Volume (%)

The experimental data are shown as means ± standard deviation and were analyzed applying one factor analysis of variance (ANOVA) performed using the Minitab 16® software. The significant differences (p < .05) between mean values of the treatments were evaluated using Tukey analysis.

4

2

3. Results and discussion 0

3.1. Particle size distribution and microstructure

0.01

3.1.1. Water-in-oil high internal emulsions The production of HIPEs using rotor-stator device (ϕ = 0.75 of disperse phase) by dropwise process allowed a slow formation of the W/O interface resulting in emulsions with small droplet size (2–4 μm) (Table 1). These results indicate that both process time and emulsifiers amount were appropriated to promote surface-active molecules adsorption onto droplet surface and establishment of interfacial film (McClements, 2015; Tadros, 2005). Water-in-oil HIPEs stabilized only with PGPR showed unimodal distribution and smaller mean droplet size (D32) than emulsions stabilized by a mixture of LEC:PGPR (Table 1 and Fig. 1A). The emulsions produced with LEC:PGPR ratio of 0.5:1.5 and 1.0:1.0 exhibited slight increase of droplet size in comparison to 0:2.0 system (Fig. 2). However, a further increase in lecithin concentration (1.5% w/w) in emulsifiers mixture led to a significant increase in mean diameter (D32). The size distribution curve displaced towards to larger droplets size and showed a broader size distribution rising lecithin concentration in the mixture. In addition, a bimodal distribution was noticed in the 1.0% LEC:1.0%PGPR and 1.5%LEC:0.5%PGPR systems (Fig. 1A). D32 and size distribution profile in emulsions produced with LEC:PGPR mixture can be explained by either molecular weight or molecular affinity of each emulsifier. Lecithin molecule is smaller than PGPR (around 4 times), although chemical affinity (solubility) of PGPR to the oil phase (low HLB value around 1–2) prevailed over the limitation imposed by PGPR high molecular weight. Hydrophilic portion of PGPR might establish hydrogen bonding with water, while the hydrophobic portion anchored into the bended chains of the sunflower oil fatty acids. The strong interactions occurring between PGPR with both aqueous and oil phases resulted in a structured interface and formation of emulsions with reduced droplet size (Michelon, Mantovani, Sinigaglia-Coimbra, de la Torre, & Cunha, 2016; Ushikubo & Cunha, 2014). On the other hand lecithin shows intermediate HLB (7–8) associated with its phospholipids composition (Fernandes et al., 2012). The lecithin used in this study was not subjected to any previous purification showing similar phospholipids content of PC, lyso-PC and PE (section 2.1). PC and lyso-PC are more suitable for production of O/W emulsion while PE for W/O emulsions. Furthermore, the enzymatic treatment

Droplet diameter (µm) Fig. 1. Droplet size distribution curves. A) W/O fresh HIPEs produced by rotorstator and B) O/W fresh emulsions produced using rotor-stator followed by high pressure homogenization. Composition of emulsifiers mixture: (eee) 0% LEC:2.0%PGPR, (e ∙ e ∙) 0.5%LEC:1.5%PGPR, (e e e) 1.0%LEC:1.0%PGPR and (∙∙∙∙∙∙) 1.5%LEC:0.5%PGPR.

could have led to a loss of one fatty acid of lyso-PC making it even more hydrophilic (Bueschelberger, Tirok, Stoffels, & Schoeppe, 2014). In view of these arguments this lecithin is an emulsifier more appropriated to form O/W emulsions than W/O emulsions which explains bigger droplet size in W/O-HIPEs (Stauffer, 2005; Wu & Wang, 2003). Thus, the quality of lecithin as a functional and technological ingredient depends on the phospholipids composition and degree of purity. 3.1.2. Oil-in-water emulsions Oil-in-water emulsions, formed after applying high pressure homogenization, showed an opposite trend in size distribution and mean droplet size when compared with W/O-HIPEs. Oil-in-water emulsions produced with the highest amount of lecithin showed unimodal distribution and the lowest D32 (Table 1 and Fig. 1B), which was confirmed with microscopy images (Fig. 3). On the other hand, the increase of PGPR proportion in emulsifiers mixture resulted in emulsions with bimodal distribution, increasing the second peak (higher size) and displacing the size distribution curve towards larger droplets size (Fig. 1B). The decrease of the mean droplet size of the O/W emulsions (Table 1) as the proportion of lecithin increased could be explained by the type of lecithin used in this work that is likely to form O/W emulsions as previously mentioned (Fernandes et al., 2012; McClements, 2015; Ushikubo & Cunha, 2014). During high pressure homogenization the W/O emulsions were subjected to intense pressure combined with cavitation, inertial and shearing forces which might have caused the breakup of big droplets to smaller ones, generating a significant increase of surface area (Floury, Desrumaux, & Lardières, 2000). The total emulsifier amount was kept fixed for all systems and might be not enough to cover the entire droplet interface created by the high-pressure process, promoting the phases 255

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

Fig. 2. Optical micrographs of W/O HIPEs freshly prepared and after 7 days of storage.

with emulsifier dynamics onto the droplet interface. The second one and probably the most significant is concerned to the droplets over packing. Compression among the droplets caused by strong mechanical forces from pressure homogenization might promote the disruption of them favouring the setting of a new continuous phase mostly based on ratio of phases composition than the polar/nonpolar balance of emulsifiers.

inversion and creating O/W emulsions. The formation of O/W emulsions as a result of energy input was confirmed by images of confocal microscopy (Fig. 4). W/O-HIPEs are characterized by a large surface area and packed droplets. In these systems, the type and amount of each emulsifier in the mixture provides a proper balance between polar and nonpolar components, which played a key role to form stable W/O emulsions. As high mechanical energy was added to these systems (high pressure homogenization) two events might have occurred. One related 256

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

Fig. 3. Optical micrographs of O/W emulsions freshly prepared and after 7 days of storage.

3.2. Rheological measurements

3.2.1. Water-in-oil high internal emulsions High internal phase emulsions show droplets closely packed favouring droplet-droplet interaction as a consequence of thin liquid layers between the droplets, which hinders the flowing of the sample and results in highly viscous emulsions. However, these interactions are weak and can break as shear stress increases explaining the shearthinning behaviour of W/O-HIPEs (Dubbelboer, Janssen, Hoogland, Zondervan, & Meuldijk, 2016; Welch, Rose, Malotky, & Eckersley,

The flow curves and rheological properties of the W/O-HIPEs and O/W emulsions are shown in Fig. 5. W/O emulsions showed shear thinning behaviour and a remarkable high viscosity. Conversely, O/W emulsions presented Newtonian behaviour and low viscosity.

257

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

Fig. 4. Confocal micrographs of the W/O-HIPEs and O/W emulsions (the area in red shows oil phase stained using the fluorescent dye Red Nile and the black shows the aqueous phase). Scale = 5 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. Kinetic stability

2006). HIPEs with the highest amount of PGPR (2% w/w) showed the smallest flow index (0.57 ± 0.01) compared to emulsions with lecithin addition (0.63–0.70), which means that the former shows a slightly more remarkable viscosity dependence of process velocity. The lower viscosity and pseudoplasticity of emulsion with higher lecithin concentration in W/O-HIPEs can be associated to the higher mean droplet size or lower surface area available for droplets interaction (Gomes, Costa, de Assis Perrechil, & da Cunha, 2016). In general, the emulsions viscosity can be tailored by the ratio between aqueous-oil phases enabling design of textures for different technological and functional applications (Gomes et al., 2016). A similar W/O emulsion with water:oil ratio of 30:70 and 60:40 with only PGPR (4% w/w) as emulsifier was evaluated and an apparent viscosity (at 3 s−1) of 160 and 1250 mPa.s, respectively, was observed (Ushikubo & Cunha, 2014). However, a dispersed phase even higher (75% w/w) led to a more viscous system (around 7800 mPa.s). Thus W/O-HIPEs approach offers the prospect of achieve viscous systems with compelling texture only by increasing the dispersed phase fraction.

Turbiscan equipment has been used to evaluate the stability of colloidal systems, including HIPEs (Gutiérrez, Matos, Benito, Coca, & Pazos, 2014; Matos, Gutiérrez, Iglesias, Coca, & Pazos, 2015). The multiple light scattering technique (backscattering profiles) allows detecting the destabilization phenomenon of emulsions before effective visual observation (Gutiérrez et al., 2014; Matos et al., 2015; TrujilloCayado, Alfaro, Muñoz, Raymundo, & Sousa, 2016). The BS measurement depends directly on the particle size since BS values decrease if the droplet size is higher than the incident wavelength. 3.3.1. Water-in-oil high internal emulsions The physical stability of W/O-HIPEs and O/W emulsions for 7 days of storage at 25 °C was evaluated (Fig. 6). In general, immediately after dropwise process in rotor-stator device, it was not observed variation of BS values as a function of the height of the measuring cell. The W/O emulsion with the highest amount of lecithin showed BS value around 80% while the other emulsions showed values closed to 90%. The lowest BS (80%) value was related to the larger droplet size of this emulsion (Table 1). A decrease of the BS values was observed after 7 days. The sample 1.5%LEC:0.5%PGPR (Fig. 6D) showed BS values of approximately 10% on the bottom-measuring cell followed by a pronounced increase (about 80%) in the top of the cell. It indicates the presence of a defined interface (serum and cream layer) and show the kinetic instability of this system during the storage (TSI = 20.35 ± 4.03). The systems stabilized by LEC:PGPR ratios of 0.5:1.5 and 1.0:1.0 showed low TSI values, 3.10 ± 0.79 and 1.30 ± 0.91 respectively. At lower lecithin concentration (Fig. 6B and C), the droplet size increased by coalescence mechanism but no signs of destabilization were observed, since the droplets were immobilized by the high viscosity of the system. Thus, the

3.2.2. Oil-in-water emulsions The high-pressure application moved the systems from “packed” W/ O emulsions (ϕ > 0.64) to concentrated O/W emulsions (0.05 < ϕ < 0.49) (McClements, 2015). The reduction of droplets amount (from 75% to 25% of disperse phase) owing to phases inversion caused a decrease of interactions among neighbouring droplets explaining the Newtonian behavior and lower viscosity observed for O/W emulsions. For these emulsions different LEC:PGPR ratio in mixtures showed no effect in Newtonian viscosity (Fig. 5B).

258

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

A)

7

Apparent viscosity (Pa.s)

measurement cell weakly affected the BS values. During storage, a creaming process took place promoted by the lower density of oil droplets, increasing BS on the top of the cell (concentrated emulsion) and decreasing on the bottom (serum phase). BS values of about 50–70% on the bottom of measuring cell is an indicative of destabilization but the gradual increase of BS values means the absence of a defined interface with no visual separation. The different emulsifier ratios did not affect TSI values (8.90–4.60) for O/W emulsions (Table 2). The viscosity of the system and concentration of each emulsifier in the mixture were the most important conditions to obtain W/O-HIPEs with enhanced stability. On the other hand, kinetic stability of O/W emulsions was highly affected by oil droplet size (Gutiérrez et al., 2014; Matos et al., 2015).

8

6 5

W/O-HIPEs

Emulsifier (% w/w) (LEC:PGPR)

k (Pa.sn)

n (-)

0.0 : 2.0 0.5 : 1.5 1.0 : 1.0 1.5 : 0.5

11.4 ± 0.3A 9.0 ± 0.2B 6.0 ± 0.3C 9.0 ± 0.2B

0.57 ± 0.01D 0.63 ± 0.01B 0.70 ± 0.01A 0.61 ± 0.01C

(mPa.s)*

7,822 ± 119A 5,404 ± 221B 2,400 ± 103D 4,505 ± 197C

*Apparent viscosity measured at shear rate of 3 s-1.

4 3

2 1

0.0%LEC:2.0%PGPR 1.0%LEC:1.0%PGPR

0 0

50

100

150

0.5%LEC:1.5%PGPR 1.5%LEC:0.5%PGPR

200

250

300

3.4. Dynamic interfacial tension and interfacial dilational rheology

Shear Rate (1/s)

B)

0.9 0.8 0.7

Shear Stress (Pa)

Interfacial tension and interfacial dilational rheology between aqueous and oil phases were studied to provide a deeper insight regarding the role of different LEC:PGPR ratios in the adsorption dynamics and interaction of these emulsifiers affecting emulsions characteristics. As aforementioned non-purified sunflower oil was used in this study. The commercial sunflower oil contains impurities such as free fatty acids, monoacylglycerol and diacylglycerol, which might exhibit surface activity, explaining the slightly decay in interfacial tension curve of the sunflower oil- water system (Fig. 7) (Dopierala et al., 2011; Gomes, Costa, & Cunha, 2018). The initial interfacial tension of the water–pure sunflower oil system was about 25 mN/m and after timeframe experiment it reached 22.5 mN/m approximately. In turn, with lecithin addition, the initial interfacial tension reduced to approximately 8.5 mN/m (Fig. 7) and values around 1.0 mN/m were quickly achieved. Polar head of lecithin is comprised by a positively charged choline group attached to a negatively charged phosphate group and this moiety is bounded to glycerol molecules. The reduction of interfacial tension is attributed to the interaction of the polar head with water molecules by hydrogen bonds, and the creation of a nonpolar interaction with the hydrocarbon chains of triacylglycerol (Ushikubo & Cunha, 2014; Whittinghill, Norton, & Proctor, 2000). Although lecithin is able to reduce interfacial tension effectively, W/O-HIPEs could not be formed with this phospholipid as single emulsifier. Phases inversion during the production of W/O emulsion was observed before complete incorporation of aqueous phase (around 60% w/w). Regarding the proportion 1.5%LEC:0.5%PGPR, the initial interfacial tension was around 12 mN/m and similar tension values to the observed only with lecithin were achieved at advanced time (3600 s). Even though PGPR increases initial interfacial tension and decreases decay rate of tension, the higher amount of lecithin seems prevail in coating the droplet interface. The lecithin quickly achieved the interface and its polar moieties were placed close to the aqueous phase while the less polar portions were faced to the oil phase. As consequence, the anchoring of PGPR onto droplet surface was less effective, which would explain the biggest droplets size observed for HIPEs. Emulsions produced with equal ratio of LEC and PGPR (1.0:1.0) exhibited a similar initial interfacial tension to the emulsion stabilized by 2.0% PGPR (17–18 mN/m). However, decay rate of tension was almost constant in timeframe experiment. This behavior distinguished among the other systems which showed a higher decay rate only in the first 100 s. As aforementioned lecithin is adsorbed leading to an abrupt drop in tension (18 to 12 mN/m) in the first seconds of assay. PGPR and remaining lecithin also reached droplet surface, which already was partially recovered by lecithin. Thus, these molecules rearrange themselves to form an interfacial film that can explain the continuous reduction of tension over time. In addition, the structured interface formed by stacking of both emulsifiers also would act as a steric barrier

1

0.6

Emulsifier (% w/w) (LEC:PGPR)

O/W emulsion

0.0 : 2.0 0.5 : 1.5 1.0 : 1.0 1.5 : 0.5

2.89 ± 0.27AB 2.48 ± 0.20B 3.00 ± 0.11 A 2.67 ± 0.06AB

(mPa.s)**

** Newtonian viscosity

0.5 0.4 0.0%LEC:2.0%PGPR

0.3

0.5%LEC:1.5%PGPR

0.2

1.0%LEC:1.0%PGPR

0.1

1.5%LEC:0.5%PGPR

0 0

50

100

150

200

250

300

Shear Rate (1/s) Fig. 5. A) Representative experimental apparent viscosity values as function of shear rate and rheological parameters (consistency index, flow behaviour index, apparent viscosity) of W/O-HIPEs. B) Representative experimental flow curves and viscosity for O/W emulsions.

extent of destabilization mechanism decreased with the addition of lecithin up to 1.0% (w/w), since an improved stability (BS ≈ 85%) was observed as compared to emulsion stabilized using only PGPR (BS = 77%). Although the PGPR-emulsion showed smaller mean droplet size its high viscosity hindered the water incorporation at more advanced times of homogenization process. Such behavior promoted the formation of some large droplets of water, which could have facilitated the increase of droplet size by coalescence justifying the more pronounced decrease of BS after 7 days of storage and high value of TSI (TSI = 12.10 ± 1.77) (Table 2). We could expect that lecithin addition would form larger droplets and less stable W/O-HIPEs. However, our results showed that appropriate LEC:PGPR ratio (1.0:1.0 and 0.5:1.5) promoted the formation of droplets as small as that stabilized with PGPR alone, but with increased kinetic stability. This finding suggests that there is a proper ratio in which PGPR and lecithin can act more efficiently on the stabilization of W/O-HIPEs. However, addition of a higher lecithin concentration (1.5% w/w) led to the formation of emulsions with low stability and increased droplet size which means that lecithin could efficiently replace PGPR until a certain limit in the emulsifier mixture. 3.3.2. Oil-in-water emulsions BS values of about 90% were observed for all fresh O/W emulsions. Due to phases inversion, the volume fraction of the dispersed phase decreased (ɸ = 0.25) and this low concentration of oil droplets in comparison to the continuous phase distributed throughout the 259

Food Research International 122 (2019) 252–262

BS (%)

P.K. Okuro, et al.

A) 100

B) 100

80

80

60

60

40

40

20

20

0

0 2

BS (%)

Fig. 6. Backscattering profiles of emulsions. W/OHIPEs: (—) fresh emulsions; (− −) after 7 days of storage. O/W emulsions produced using rotor-stator followed by high pressure homogenization process: (∙∙∙∙∙) fresh emulsions; (∙∙—∙∙) after 7 days of storage. Emulsions formulated with emulsifiers mixture: A) 0% LEC:2.0% PGPR, B) 0.5% LEC:1.5% PGPR, C) 1.0% LEC:1.0% PGPR and D) 1.5% LEC:0.5% PGPR.

8

14

20

26

32

38

C) 100

D) 100

80

80

60

60

40

40

20

20

0

2

8

14

20

26

32

38

2

8

14

20

26

32

38

0 2

8

14

20

26

32

38

Height (mm)

Height (mm)

around 9 mN/m. In turn, lecithin as single emulsifier revealed the less rigid interface with the lowest complex modulus value. Interestingly LEC:PGPR mixtures (0.5:1.5 and 1.0:1.0 ratio) yielded highly viscoelastic interface which increased viscoelastic modulus over time which can be attributed to positive interaction between these emulsifiers. Finally, in the LEC:PGPR ratio of 1.5:0.5, lecithin behavior predominated over PGPR leading to lower values and a drop of complex modulus over time which is in agreement with results from interfacial tension. It is known that the coalescence of the droplets would be avoided by the viscoelastic characteristic of the interface, shown by the relatively high complex modulus values. In fact the relationship between complex viscoelastic modulus and the stability of the emulsions was previously addressed by other studies (Gomes et al., 2018; Ushikubo & Cunha, 2014). The more viscoelastic interfacial layer observed for LEC:PGPR mixtures (0.5:1.5 and 1.0:1.0 ratios) led to an enhanced kinetic stability of emulsions. Therefore the interface characteristics corroborated the results disclosed by backscattering values (Fig. 6). The presence of lecithin at certain ratios could increase the dilational modulus strengthening the interfacial film stabilized by PGPR because of the reinforcement of lecithin. This positive interaction could explain the more efficient stabilizing mechanism. LEC/PGPR mixtures turned to be good emulsifiers for W/O-HIPEs impacting directly on the kinetic stability as well in other emulsion properties. The best LEC:PGPR ratios were 0.5:1.5 and 1.0:1.0. The hypothesis is that the interface would be firstly covered by lecithin (first layer) leading to a pronounced decrease of interfacial tension. The different phospholipids will be displayed in the different phases according their polarity. After that, the PGPR could be attached to the droplet interface forming an external layer between lecithin and oil establishing a reinforced interfacial layer with alternate composition of lecithin and PGPR.

Table 2 Turbiscan stability index (TSI) values for W/O-HIPEs and O/W emulsions aged for 7 days. Emulsifier (% w/w) (Lecithin: PGPR)

0: 2.0 0.5: 1.5 1.0: 1.0 1.5: 0.5

W/O-HIPEs

O/W emulsion

TSI

TSI

12.10 ± 1.77 3.10 ± 0.79C 1.30 ± 0.91C 20.35 ± 4.03

B

A

8.37 7.73 8.90 4.60

± ± ± ±

4.55 0.78 1.48 1.15

A A A A

Different letters indicate significant difference at p < .05 in the same column.

contributing to the kinetic stability. An additional reduction of lecithin in the mixture (0.5%LEC:1.5% PGPR) resulted in a tension curve similar to the system with 2%PGPR suggesting that the adsorbed interfacial layer was comprised mainly by PGPR molecules. In this configuration lecithin would work as a fillerspace of PGPR molecules, acting as co-surfactant, reducing the required time to achieve equilibrium tension. The structured layer formed with this emulsifiers mixture ratio can be correlated with its good kinetic stability. Finally, it was observed a reduction of initial interfacial tension from 17 mN/m reaching values around 3 mN/m for monocomponent PGPR system. PGPR can form a structured interface in W/O emulsions promoting steric hindrance that along with the formation of emulsion with small droplet size contributed for a good kinetic stability of PGPRemulsions (Benichou, Aserin, & Garti, 2001; Ushikubo & Cunha, 2014). However in HIPEs other variables such as viscosity of emulsion and volume fraction of dispersed phase can also affect emulsions stability (McClements, 2015). The complex viscoelastic modulus of the water-sunflower oil with lecithin, PGPR or their corresponding mixtures are presented in Fig. 7 (B). Emulsions stabilized only by PGPR showed a slight decrease of viscoelastic modulus during timeframe experiment reaching values

4. Conclusion High-energy input from high-pressure homogenization set the 260

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

58017-5, 2009/50591-0 and 2011/06083-0). Matheus Augusto Adame thanks to CNPq/PIBIC (122956-2015-0). Paula Kiyomi Okuro thanks FAPESP (grant #2015/24912-4; 2016/10277-8; 2018/20308-3 São Paulo Research Foundation (FAPESP)) and CNPq (409314/2017-0), Andresa Gomes Brunassi thanks CNPq (140705/2015-5) and Ana Leticia Rodrigues Costa Lelis thanks CNPq (140710/2015-9) for the PhD assistantship; Rosiane Lopes Cunha thanks CNPq (307168/2016-6) for the productivity grant. The authors also thank the access to Zeiss confocal microscope LSM 780 NLO and assistance provided by the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas; INFABIC is co-funded by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) (08/57906-3) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) (573913/2008-0).

A) 27

Interfacial tension (mN/m)

24 21 18 15 12

9 6 3 0 0

600

1200 1800 2400 3000 3600 Time (s)

References

Complex Modulus (mN/m)

B)

21 Alicja, M., Fernando, A., Riccardo, C., Alessandro, D. D., Birgit, D., Jose, F. M., ... Claude, L. (2017). Re-evaluation of polyglycerol polyricinoleate (E 476) as a food additive. EFSA Journal, 15(3), 4743. Bai, L., Huan, S., Li, Z., & McClements, D. J. (2017). Comparison of emulsifying properties of food-grade polysaccharides in oil-in-water emulsions: Gum arabic, beet pectin, and corn fiber gum. Food Hydrocolloids, 66, 144–153. Belayneh, H. D., Wehling, R. L., Cahoon, E., & Ciftci, O. N. (2018). Lipid composition and emulsifying properties of Camelina sativa seed lecithin. Food Chemistry, 242, 139–146. Benichou, A., Aserin, A., & Garti, N. (2001). Polyols, high pressure, and refractive indices equalization for improved stability of W/O emulsions for food applications. Journal of Dispersion Science and Technology, 22(2–3), 269–280. Bueschelberger, H.-G., Tirok, S., Stoffels, I., & Schoeppe, A. (2014). Lecithins Emulsifiers in food technology. John Wiley & Sons, Ltd21–60. Dashiell, G. L. (1989). Lecithin in food processing applications. In B. F. Szuhaj (Ed.). Lecithins: Sources, manufacture & uses (pp. 218–219). . Dedinaite, A., & Campbell, B. (2000). Interactions between mica surfaces across triglyceride solution containing phospholipid and polyglycerol Polyricinoleate. Langmuir, 16(5), 2248–2253. Dickinson, E. (2015). Colloids in food: Ingredients, structure, and stability. Annual Review of Food Science and Technology, 6, 211–233. Dopierala, K., Javadi, A., Krägel, J., Schano, K. H., Kalogianni, E. P., Leser, M. E., & Miller, R. (2011). Dynamic interfacial tensions of dietary oils. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 382(1–3), 261–265. Dubbelboer, A., Janssen, J. J. M., Hoogland, H., Zondervan, E., & Meuldijk, J. (2016). Pilot-scale production process for high internal phase emulsions: Experimentation and modeling. Chemical Engineering Science, 148, 32–43. Fernandes, G. D., Alberici, R. M., Pereira, G. G., Cabral, E. C., Eberlin, M. N., & BarreraArellano, D. (2012). Direct characterization of commercial lecithins by easy ambient sonic-spray ionization mass spectrometry. Food Chemistry, 135(3), 1855–1860. Floury, J., Desrumaux, A., & Lardières, J. (2000). Effect of high-pressure homogenization on droplet size distributions and rheological properties of model oil-in-water emulsions. Innovative Food Science and Emerging Technologies, 1(2), 127–134. Gomes, A., Costa, A. L. R., & Cunha, R. L. (2018). Impact of oil type and WPI/tween 80 ratio at the oil-water interface: Adsorption, interfacial rheology and emulsion features. Colloids and Surfaces B: Biointerfaces, 164, 272–280. Gomes, A., Costa, A. L. R., de Assis Perrechil, F., & da Cunha, R. L. (2016). Role of the phases composition on the incorporation of gallic acid in O/W and W/O emulsions. Journal of Food Engineering, 168, 205–214. Guiotto, E. N., Cabezas, D. M., Diehl, B. W. K., & Tomás, M. C. (2013). Characterization and emulsifying properties of different sunflower phosphatidylcholine enriched fractions. European Journal of Lipid Science and Technology, 115(8), 865–873. Gülseren, İ., & Corredig, M. (2012). Interactions at the interface between hydrophobic and hydrophilic emulsifiers: Polyglycerol polyricinoleate (PGPR) and milk proteins, studied by drop shape tensiometry. Food Hydrocolloids, 29(1), 193–198. Gülseren, İ., & Corredig, M. (2014). Interactions between polyglycerol polyricinoleate (PGPR) and pectins at the oil–water interface and their influence on the stability of water-in-oil emulsions. Food Hydrocolloids, 34(0), 154–160. Gutiérrez, G., Matos, M., Benito, J. M., Coca, J., & Pazos, C. (2014). Preparation of HIPEs with controlled droplet size containing lutein. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 442, 111–122. Jafari, S. M., Assadpoor, E., He, Y., & Bhandari, B. (2008). Re-coalescence of emulsion droplets during high-energy emulsification. Food Hydrocolloids, 22(7), 1191–1202. Joshi, A., Paratkar, S. G., & Thorat, B. N. (2006). Modification of lecithin by physical, chemical and enzymatic methods. European Journal of Lipid Science and Technology, 108, 363–373. Kralova, I., Sjöblom, J., Kralova, I., & Sjöblom, J. (2009). Surfactants used in food industry: A review. Vol. 30, 1363–1383. Lett, A. M., Norton, J. E., & Yeomans, M. R. (2016). Emulsion oil droplet size significantly affects satiety: A pre-ingestive approach. Appetite, 96, 18–24. Lucassen, J., & Van Den Tempel, M. (1972). Dynamic measurements of dilational properties of a liquid interface. Chemical Engineering Science, 27(6), 1283–1291. Márquez, A. L., Medrano, A., Panizzolo, L. A., & Wagner, J. R. (2010). Effect of calcium

18 15 12 9 6 3 0 0

5 10 15 20 25 30 35 40 45 50 Time (min)

Fig. 7. A) Dynamic interfacial tension and B) interfacial dilational rheology of the systems composed by water-pure sunflower oil ( ) or emulsifiers mixture: ( ) 0% LEC:2.0% PGPR, ( ) 0.5% LEC:1.5% PGPR, ( )1.0% LEC:1.0% PGPR and ( ) 1.5% LEC:0.5% PGPR and ( ) 2.0% LEC:0% PGPR.

formation of O/W emulsions from W/O-HIPEs since phases inversion. The O/W emulsions features were affected by the LEC:PGPR ratio in emulsifier mixture. In fact, W/O-HIPEs were formed from process with lower energy demand such as dripping. The use of lecithin as alternative for partial replacement of PGPR in W/O-HIPEs was successfully performed until 50% of the emulsifier-required amount. The proper LEC:PGPR ratios of emulsifiers were 0.5:1.5 and 1.0:1.0 resulting in emulsions with good kinetic stability attributed to the formation of an enhanced viscoelastic interfacial layer. This finding is relevant especially considering that lecithin used in this study was non-purified, which is usually associated with lower cost. In addition, different physicochemical and structural characteristics can arise as a result of the partial replacement of PGPR by lecithin in emulsion. The mixtures might be applied without compromise the kinetic stability of emulsions allowing to manipulate and create new textures aiming food applications. Thus, hybrid LEC:PGPR- based W/O-HIPEs (with reduction of semi-synthetic ingredient) could be tailored by the ratio LEC:PGPR in emulsifier mixture. Finally, in view of different technological, functional and nutritional approaches the use of lecithin and PGPR mixtures could be employed to form stable and versatile W/O-HIPEs. Moreover the comparative approach of the same emulsifier mixture in analogue O/W emulsion highlighted the rationality involving ingredient engineering for each system depending its characteristics.

Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (DEA/ FEA/PROEX) and FAPESP (EMU2009/54137-1, 2004/08517-3, 2007/ 261

Food Research International 122 (2019) 252–262

P.K. Okuro, et al.

Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34(0), 145–153. Welch, C. F., Rose, G. D., Malotky, D., & Eckersley, S. T. (2006). Rheology of high internal phase emulsions. Langmuir, 22(4), 1544–1550. Whittinghill, J. M., Norton, J., & Proctor, A. (2000). Stability determination of soy lecithin-based emulsions by Fourier transform infrared spectroscopy. Journal of the American Oil Chemists' Society, 77(1), 37–42. Wilson, R., & Smith, M. (1998). Human studies on polyglycerol Polyricinoleate (PGPR). Food and Chemical Toxicology, 36(9–10), 743–745. Wu, Y., & Wang, T. (2003). Soybean lecithin fractionation and functionality. JAOCS, 80(4), 319–326. Yang, T., Zheng, J., Zheng, B.-S., Liu, F., Wang, S., & Tang, C.-H. (2018). High internal phase emulsions stabilized by starch nanocrystals. Food Hydrocolloids, 82, 230–238. Yokota, D., Moraes, M., & Pinho, S. C. (2012). Characterization of lyophilized liposomes produced with non-purified soy lecithin: A case study of casein hydrolysate microencapsulation. Brazilian Journal of Chemical Engineering, 29(2), 325–335. Zeng, T., Wu, Z.-l., Zhu, J.-Y., Yin, S.-W., Tang, C.-H., Wu, L.-Y., & Yang, X.-Q. (2017). Development of antioxidant Pickering high internal phase emulsions (HIPEs) stabilized by protein/polysaccharide hybrid particles as potential alternative for PHOs. Food Chemistry, 231, 122–130. Zhang, B., Zhang, J., Liu, C., Peng, L., Sang, X., Han, B., ... Yang, G. (2016). High-internalphase emulsions stabilized by metal-organic frameworks and derivation of ultralight metal-organic aerogels. Scientific Reports, 6.

salts and surfactant concentration on the stability of water-in-oil (w/o) emulsions prepared with polyglycerol polyricinoleate. Journal of Colloid and Interface Science, 341(1), 101–108. Matos, M., Gutiérrez, G., Iglesias, O., Coca, J., & Pazos, C. (2015). Characterization, stability and rheology of highly concentrated monodisperse emulsions containing lutein. Food Hydrocolloids, 49, 156–163. McClements, D. J. (2015). Food emulsions: Principles, practices, and techniques (3rd ed.). Taylor & Francis. Michelon, M., Mantovani, R. A., Sinigaglia-Coimbra, R., de la Torre, L. G., & Cunha, R. L. (2016). Structural characterization of β-carotene-incorporated nanovesicles produced with non-purified phospholipids. Food Research International, 79, 95–105. Prichapan, N., McClements, D. J., & Klinkesorn, U. (2017). Influence of rice bran stearin on stability, properties and encapsulation efficiency of polyglycerol polyricinoleate (PGPR)-stabilized water-in-rice bran oil emulsions. Food Research International, 93, 26–32. Stauffer, C. E. (2005). Emulsifiers for the food industry. Bailey's industrial oil and fat products. John Wiley & Sons, Inc.. Tadros, T. F. (2005). Applications of surfactants in emulsion formation and stabilisation Applied Surfactants. Wiley-VCH Verlag GmbH & Co. KGaA115–185. Trujillo-Cayado, L. A., Alfaro, M. C., Muñoz, J., Raymundo, A., & Sousa, I. (2016). Development and rheological properties of ecological emulsions formulated with a biosolvent and two microbial polysaccharides. Colloids and Surfaces B: Biointerfaces, 141, 53–58.

262