Optimization of Multiple W1/O/W2 Emulsions Processing for Suitable Stability and Encapsulation Efficiency

Citation: Felix, M.; Guerrero, A.; Carrera-Sánchez, C. Optimization of Multiple W1/O/W2Emulsions Processing for Suitable Stability and Encapsulation Efficiency. Foods 2022, 11, 1367. https://doi.org/10.3390/ foods11091367 Academic Editor: Lars Nilsson Received: 1 April 2022 Accepted: 6 May 2022 Published: 9 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article Optimization of Multiple W1/O/W2Emulsions Processing for Suitable Stability and Encapsulation Efficiency Manuel Felix * , Antonio Guerrero and Cecilio Carrera-Sánchez Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011 Sevilla, Spain; aguerrer[email protected] (A.G.); [email protected] (C.C.-S.) *Correspondence: [email protected] Abstract: Double emulsions are a type of multiple emulsions, which can be defined as a multicompartmentalized system where the droplets are dispersed into the continuous phase containing other emulsions. Although double food-grade emulsions have been manufactured, there is a lack of scientific background related to the influence of different processing conditions. This work analyses the influence of processing variables in (W 1 /O/W 2 ) double emulsions: passes through the valve homogenizer, pressure applied, lipophilic emulsifier concentration, the ratio between the continuous phase (W 2 ) and the primary emulsion (W 1 /O), and the incorporation of xanthan gum (XG) as a stabilizer. The results obtained show that these emulsions can be obtained after selecting suitable processing conditions, making them easily scalable in industrial processes. In terms of droplet size distribution, the input of higher energy to the system (20 MPa) during emulsification processing led to emulsions with smaller droplet sizes (D 3,2 ). However, more monodispersed emulsions were achieved when the lowest pressure (5 MPa) was used. As for the number of passes, the optimal (emulsions more monodispersed and smaller droplet sizes) was found around 2–3 passes, regardless of the valve homogenizer pressure. However, emulsions processed at 20 MPa involved lower encapsulation efficiency (E E ) than emulsions processed at 5 MPa (87.3 ± 2.3 vs. 96.1 ± 1.8, respectively). The addition of XG led to more structured emulsions, and consequently, their kinetic stability increased. The results obtained indicated that a correct formulation of these W 1 /O/W 2 double emulsions allowed the optimal encapsulation of both hydrophilic and lipophilic bioactive compounds. Thus, the development of food matrices, in the form of multiple emulsions, would allow the encapsulation of bioactive compounds, which would result in the development of novelty food products. Keywords: droplet size distribution; emulsification; multiple emulsions; polyglycerol polyricinoleate (PGPR) 1. Introduction Functional food products are based on the presence of functional ingredients (bioactive compounds) in the food matrix; the possibility of developing this new generation of food products requires the employment of strategies able to preserve the desired components during their storage and after their intake thorough the intestinal tract [ 1 ]. One approach for the development of these functional foods includes the use of current technologies applied to emulsion-based products, making them a reliable alternative to the current food market [ 2 ]. In this way, the most versatile path to modify the composition of food arises from the possibility of introducing bioactive ingredients during its preparation [ 3 ]. Based on the solubility of these ingredients, they could be classified into two big families: lipophilic and hydrophilic compounds [ 4 ]. The former ingredients (e.g., carotenoids, E-vitamin, etc.) are soluble in the oil phase of conventional emulsions, although their encapsulation in nanoemulsion-based systems has been demonstrated to improve not only the physical stability but also their absorption after intake [ 3 ]. However, hydrophilic components (e.g., polyphenols, peptides, etc.) cannot be included in the continuous phase Foods 2022,11, 1367. https://doi.org/10.3390/foods11091367 https://www.mdpi.com/journal/foods Foods 2022,11, 1367 2 of 17 of conventional emulsions since their biodisponibility is reduced after digestion [ 5 ]. An alternative for introducing these bioactive components in food matrices is to include them in the primary emulsion of double emulsions, protecting them from chemical changes during digestion [6,7]. In this context, the development of double emulsions as food matrices opens up new possibilities in the design and development of functional foods [ 8 ]. These food matrices could be used as carriers of water-soluble bioactive components. However, technological strategies must be applied to adapt current food processing methods in the development of this type of emulsion, optimizing the presence of bioactive compounds [8]. Double emulsions are a type of multiple emulsion, which can be defined as a multicompartmentalized system where the droplets disperse into the continuous phase containing other emulsions [ 9 ]. These systems are characterized by the coexistence of oil-in-water (O/W) and water-in-oil (W/O) emulsions, in which the dispersed phase (in the form of droplets) is the continuous phase of the primary emulsion (which means that it has smaller droplets of the other phase disperse into it) [ 10 ]. Thus, the most common case is waterin-oil-in-water emulsions (W 1 /O/W 2 ), although those of oil-in-water-in-oil (O 1 /W/O 2 ) can also be used in specific applications (i.e., they exhibit a fine texture, showing a smooth touch upon application) [ 6 , 11 ]. Once produced, double emulsions are subjected to several coalescence and diffusion phenomena that consequently impact product properties, such as texture or encapsulation performance [12]. The use of multiple (double) emulsions in food matrices brings unexplored opportunities to introduce bioactive compounds in food products and, consequently, in human consumption habits. Although double food-grade emulsions have been already manufactured, there is a lack of scientific background related to the influence of different processing conditions. The general objective of this work is to analyze the influence of processing variables that affect the development of double emulsion (W 1 /O/W 2 ). The encapsulation efficiency (E E , parameter to be optimized) was assessed by tartrazine, whose use as a marker was initially proven. The different variables studied were: passes through the valve homogenizer, pressure applied, lipophilic emulsifier (polyglycerol polyricinoleate, PGPR), the ratio between the continuous phase (W 2 ) and the primary emulsion (W 1 /O), and the incorporation of a hydrocolloid (xanthan gum) as a stabilizer of the double emulsions generated. 2. Materials and Methods 2.1. Materials The lipophilic surfactant polyglycerol polyricinoleate (PGPR) was supplied by Campobetica (Malaga, Spain). This emulsifier was used for the stabilization of primary emulsions (W 1 /O). BiPro ® whey protein isolate (WPI), supplied by AGROPUR (Longueuil, QC, Canada), was the emulsifier used for the stabilization of secondary emulsions (W 1 /O/W 2 ). Xanthan from Xanthomonas campestris was provided by Merck (Branchburg, NJ, USA). Chemical reagents (i.e., HCl, NaOH, NaCl, Tartrazine and Trizma) were purchased from Sigma–Aldrich company (St. Louis, MO, USA). The oil used (sunflower oil) was purchased from a local market. Emulsions were prepared using deionized water. 2.2. Methods 2.2.1. Preparation of W1/O/W2Double Emulsions Firstly, the primary W 1 /O emulsion was prepared. For these emulsions, the aqueous phase was stabilized at pH 7.0 using Trizma (50 mM). NaCl was added for ionic-strength control (50 mM), and tartrazine was used as a marker (0.06 wt.%). This aqueous phase (W 1 ) was dispersed into the continuous oily phase, which contained PGPR (1, 3 or 5 wt.%). The W 1 /O ratio of these primary emulsions was, in all cases, 20/80. This primary emulsion was prepared by applying high energy to obtain small-sized droplets. Thus, the oil and the W 1 phase were blended and mixed using a rotor-stator homogenizer (Utraturrax ® T-25, IKA, Staufen, Germany) at 8000 rpm. Subsequently, the emulsion obtained was passed Foods 2022,11, 1367 3 of 17 through a high-pressure homogenizer at 50 MPa (Emulsiflex 2000-CS, Avestin, Ottawa, ON, Canada). Once primary emulsions were prepared, secondary emulsions were generated by mixing the primary emulsion with an aqueous WPI solution (0.5 wt.%) at pH 7, W 2 . In order to adjust the osmotic pressure balance between the aqueous phases W 1 and W 2 , NaCl was also added to this outer aqueous phase. Soft processing conditions (i.e., low energy input) were used in this case to avoid the breakdown of primary emulsions. The W 1 /O primary emulsion was mixed with the outer phase (W 2 ) using the same rotor-stator mixer, but at 1000 rpm. These pre-emulsions were also passed through the Emulsiflex valve homogenizer, selecting, in this case, 5 and 20 MPa. The (W 1 /O)/W 2 ratios of multiple emulsions were 10/90, 25/75 and 40/60. These double emulsions were prepared according to the method proposed by Lynch [ 13 ]. Moreover, the influence of the addition of xanthan gum (XG) as a stabilizer was also tested. These emulsions were prepared following the same procedure described above with a (W 1 /O)/W 2 ratio of 40/60 (processed in absence of XG). Once emulsions were generated (using 5 wt.% PGPR according to the highest EE value), XG dissolved into W 2 was added up to reach the (W 1 /O)/W 2 ratio of 25/75. XG solution was dispersed into (W 1 /O)/W 2 emulsions by soft magnetic stirring. The final XG concentration studied was 0.125 and 0.25 wt.%. Supplementary Table S1 summarizes the systems performed in this research. 2.3. Characterisation of Emulsions 2.3.1. Droplet Size Distribution Droplet size distribution (DSD) of the double emulsions (W 1 /O/W 2 ) was determined with the particle size analyzer based on laser diffraction Mastersizer 2000 (Malvern, UK). To facilitate the rupture of possible flocs, 0.5 mL of emulsion samples were diluted in a 1% SDS (sodium dodecyl sulfate) solution. Immediately after this, the droplet size distribution of the deflocculated systems was determined [ 14 ]. The mean Sauter diameter (D 3,2 ) and volumetric diameter (D4,3) were calculated according to Equations (1) and (2): D3,2 =∑nid3 i ∑nid2 i (1) D4,3 =∑nid4 i ∑nid3 i (2) where niis the number of droplets that have didiameter. The span parameter (Equation (3)) was calculated to analyze the DSD profiles: Span =d(90)−d(10) d(50)(3) where d(90) , d(10) and d(50) refer to the 10, 50 and 90 volume percentile of droplets with diameters smaller or equal to these values. The Flocculation Index (FI, Equation (4)) was calculated to study the flocculation phenomena occurring during emulsion storage: FI (%)=   D4,3 −D4,3 (SDS)    D4,3 ×100 (4) where D4,3 and D4,3 (SDS)are the volumetric diameter without and with SDS solution. 2.3.2. Rheological Properties Linear viscoelastic properties were analyzed by means of small amplitude oscillatory shear (SAOS) tests using the AR-2000 rheometer (TA Instruments, New Castle, DE, USA). All measurements were carried out within the linear viscoelastic range (LVR), which was determined by means of strain sweep tests. These tests were performed at 1 Hz and from Foods 2022,11, 1367 4 of 17 0.01 to 10% strain. Mechanical spectra were obtained from frequency sweep tests (from 0.1 to 30 Hz) at a constant strain (1%). These tests were carried out using a low inertia aluminum smooth plate (60 mm) at a gap of 1 mm. The temperature was fixed at 25 ◦ C by a peltier system 2.3.3. Emulsion Stability The overall stability of emulsions was assessed using a vertical scan analyzer Turbiscan MA 2000 (Formulaction, Toulouse, France) through backscattering measurements of a pulsed light source ( λ = 850 nm) as a function of the height of the cylindrical glass tube containing the sample [ 15 ]. Emulsions were stored at 5 ◦ C and measured at room temperature. Then, the profiles of backscattering of light from emulsions ( ∆ BS/100%) versus the height were plotted as a function of storage time. 2.4. Encapsulation Capacity 2.4.1. Measurement of Marker Concentration in the Outer Aqueous Phase (W2) One of the most important steps in the determination of the marker concentration is to ensure that the concentration determined for the marked is not affected by the presence of the protein used, the processing conditions or the storage time. In this section, experiments were carried out to ensure the correct measurement of the marker in the final double emulsions [16]. The first determination consisted of checking the correct determination of the marker (tartrazine) in the presence of a WPI solution. The marker was dissolved in Trizma buffer ( 50 mM , pH 7) at the same concentration as it was added to the inner aqueous phase (W 1 ) in the double emulsion (0.06 wt.%). Subsequently, this solution was added to the outer aqueous phase W 2 at the same ratio as in the final double emulsions. The W 2 phase contained 0.5 wt.% WPI protein since this is the protein concentration used in the W 2 of the final double emulsions. W 1 + W 2 were mixed at room temperature for 30 min using a magnetic stirrer. The concentration of the marker in the overall solution was calculated by measuring the absorbance at 435 nm of the corresponding solution using a spectrophotometer (Thermo Scientific, Waltham, MA, USA), using the WPI in Trizma buffer as the blank reference. Tartrazine solution was also added to a Trizma buffer solution (without WPI) and treated, but in this case, using the buffer solution as the blank reference. The value of absorbance obtained was interpolated in a calibration curve obtained after dissolving different known concentrations of the marker in Trizma buffer at pH 7 (W1). Measurements were carried out on the same day of W 1 + W 2 mixing and after 7 storage days in an oven at 45 ◦ C. The marker measurable concentration ( MCm(%) ) in the WPI solution was calculated according to the Equation (5): MCm(%)=CmWPI Cmb ×100 (5) where CmWPI is the concentration of the marker measured in the W 1 + W 2 (with WPI) mixtures and Cmb is the concentration of the marker according to the dilutions carried out (without WPI). 2.4.2. Measurement of the Recovery Yield (Ry) of Marker The recovery yield is defined as the concentration of marker used, which is present in the aqueous phase recovered after separating the cream phase and an aqueous phase by centrifugation. The percentage of recovery yield is relative to the concentration of marker in the outer aqueous phase after emulsion preparation [16]. For these measurements, a conventional oil-in-water emulsion (O/W 2 ) was prepared using the same oil phase (O) and the same outer aqueous phase (W2) as in the W1/O/W2 double emulsion (ratio 10/90). The oil phase (O) was gradually added to the aqueous phase (W 2 ) while they were mixing using an Ultraturrax ® homogenizer (IKA, Staufen, Germany) at 9500 rpm and 1 min. Subsequently, the pre-emulsion was passed through a Foods 2022,11, 1367 5 of 17 high-pressure homogenizer (Emulsiflex 2000-CS, Avestin, Ottawa, ON, Canada). These O/W 2 conventional emulsions were diluted in the W 1 phase (reaching the ratio eventually used in W 1 /O/W 2 double emulsions). The W 1 contained either (i) the marker at 0.06 wt.% for the determination of marker recovery or (ii) without the marker for the sample blank. According to Dickinson et al. [ 17 ], this procedure ensured that the marker was completely present in the outer aqueous phase. Consequently, this emulsion can be used as the standard where 100% of the marker was present in the outer aqueous phase (W 2 ). The determination of aqueous phase color requires a transparent medium. Thus, an aliquot of the emulsions was diluted with the W 1 phase (1:1), the mixture was homogenized by a soft stirring and, subsequently, it was separated by centrifugation (15,000 × gat 4 ◦ C for 30 min) in a cream phase (which contained the oil droplets) and an aqueous phase. The aqueous phase was filtered (0.2 µ m syringe microfilter), and the concentration of the marker presented in the resulting filtered aqueous phase was calculated by the measurement of the absorbance at 435 nm in a spectrophotometer (Thermo Scientific) using the subnatant obtained from O/W 2 emulsion with added Trizma buffer without the added marker as a reference blank. The value of the absorbance obtained was compared with the concentration of the marker in the outer aqueous phase (which depends on the ratio (W 1 /O)/W 2 ). The recovery yield can be calculated according to Equation (6) [16]: Ry (%)=CmOAP(1,7) Cmb ×100 (6) where CmOAP(1,7) was the concentration of the marker used present in the outer aqueous phase (recovered after centrifugation) after 1 and 7 days of emulsion aging. Cmb was the concentration of the marker used in the outer aqueous phase. This latter concentration was determined by an equivalent dilution of the aqueous marker solution with Trizma buffer as occurred in the emulsion. The concentration of the marker present in the outer aqueous phase was calculated on the same day of emulsion preparation and 7 days later to determine the influence of aging on the Ry parameter. 2.4.3. Encapsulation Efficiency (EE) Measurement The E E of the double W 1 /O/W 2 emulsion was calculated by the determination of the concentration of the marker in the outer aqueous phase (W 2 ) just after the preparation of the emulsions. The E E of double W 1 /O/W 2 emulsions was prepared according to the experimental procedure indicated in Section 2.2.1. Once double emulsions were prepared, they were subjected to the phase separation explained in Section 2.4.2. Double emulsions were prepared with the marker (tartrazine) diluted in the W 1 and in the absence of the marker to obtain the blank for the measurement of the absorbance of the aqueous phase. The concentration of the marker in the outer aqueous phase (W 2 ) was measured by the same method as described above for measuring the recovery yield but replacing the O/W 2 emulsion with the W 1 /O/W 2 emulsion. The E E (%) was calculated with respect to the concentration of the marker present in the aqueous phase obtained after emulsion separation (W 1 + W 2 ) according to Equation (7): EE(%)=100 −CmOAP Cmb ×100 (7) where CmOAP is the marker concentration measured in the aqueous phase (which has been recovered from the double emulsions), Cmb is the marker concentration initially added to the inner aqueous phase. The E E was calculated on days 0 and 15 to determine the influence of storage time on this parameter. Foods 2022,11, 1367 6 of 17 2.5. Statistical Analysis Emulsions were prepared in duplicate. Three replicates were carried out for each measurement. Significant differences were determined by performing ANOVA tests using STATGRAPHICS Centurion XVIII software version number (Statgraphics Tecnologies Inc., The Plains, VA, USA). 3. Results and Discussion 3.1. Evaluation of Tartrazine as a Marker for Double Emulsions Stabilized by WPI The effectiveness of tartrazine as a marker for encapsulating hydrophilic compounds in W 1 /O/W 2 double emulsions must be addressed before any experiment. To this end, it is essential to identify a marker added to the internal aqueous phase (W 1 ), whose concentration in the outer aqueous phase (W 2 ) can be determined after double emulsion preparation. Moreover, changes in the concentration of the marker should not be elucidated in the outer aqueous phase during double emulsion storage [16]. Table 1shows the marker measurable concentration ( MCm ) and recovery yield (Ry) for the marker used in this work (tartrazine) in the aqueous phase recovered when 100% of the marker is in the outer aqueous phase (W 2 ). Table 1indicates that the marker used can be accurately measured in the external aqueous phase (W 2 ) of the double W 1 /O/W 2 emulsion by measuring the absorbance of the outer aqueous phase following the procedure indicated in Section 2.4 since the marker concentration measurable (MC m ) reached 100 ±0.5%. Moreover, there are no changes in the concentration of tartrazine after 7 days of external phase (W2) storage containing tartrazine at 45 ◦C (MCm= 98.8 ±0.2%). Table 1. Marker concentration measurable (MC m ) and recovery yield (Ry) for the marker (tartrazine) in the aqueous phase recovered standard where 100% of the marker is in the outer aqueous phase (W2). Different letters within a row indicate significant differences (p< 0.05). Day MCm(%) Ry (%) 0 100.0 ±0.5 a95.5 ±1.5 a 798.8 ±0.2 b98.9 ±0.2 a As for the recovery yield (Ry), this table also reveals that the marker selected (tartrazine) can be used for the evaluation of this parameter since the 95.5 ± 1.5% tatrazine remains in the inner aqueous phase (W 1 ) the same day emulsion preparation, and 98.9 ±0.2% after 7 days, where no significant differences were found (p< 0.05). These results also show that the tartrazine marker does not preferentially migrate with the cream phase formed after emulsion centrifugation. The high level of marker present in the recovered aqueous phase after centrifugation suggests that tartrazine is equally distributed throughout the aqueous phase of the entire emulsion. These results indicate that tartrazine does not associate with WPI and that this marker can be used for the evaluation of emulsion efficiency (E E ) for the double W 1 /O/W 2 emulsion generated hereinafter. These parameters were more suitable than the values found when methylene blue and B 12 vitamin were used as markers in double emulsions stabilized by sodium caseinate [16]. 3.2. Processing Conditions, Optimization Passes and Valve Homogenizer Pressure Droplet Size Distribution Measurements The ratio 25/75 was used for double emulsions ((W 1 /O)/W 2 ) during the optimization of the number of passes through the high-pressure homogenizer (from 1 to 5). The homogenization pressures evaluated were 5 and 20 MPa. The PGPR concentration used for these emulsions was 5 wt.%. Figure 1shows the droplet size distribution (DSD) of the W 1 /O/W 2 emulsions obtained after processing the secondary emulsion at 5 MPa (A) and 20 MPa (B) as a function Foods 2022,11, 1367 7 of 17 of emulsion passes through the valve homogenizer. All measurements plotted were carried out in the presence of 1% SDS (deflocculating agent) to avoid DSD influenced by the bridging flocculation phenomena [ 14 ]. When the homogenization pressure was 5 MPa (Figure 1A), unimodal DSD profiles were obtained in all cases; however, the distribution becomes narrower as emulsion passes increased. Thus, the distributions always started around 2 µ m; however, the highest droplets moved from c.a. 40 to 20 µ m, where the reduction of droplet sizes most important took place after passes 1 and 2. This reduction in droplet size can be expected since there is an increase in the energy input during emulsion processing as the number the passes increases [18]. Foods 2022, 11, x FOR PEER REVIEW 7 of 18 3.2. Processing Conditions, Optimization Passes and Valve Homogenizer Pressure Droplet Size Distribution Measurements The ratio 25/75 was used for double emulsions ((W1/O)/W2) during the optimization of the number of passes through the high-pressure homogenizer (from 1 to 5). The homogenization pressures evaluated were 5 and 20 MPa. The PGPR concentration used for these emulsions was 5 wt.%. Figure 1 shows the droplet size distribution (DSD) of the W1/O/W2 emulsions obtained after processing the secondary emulsion at 5 MPa (A) and 20 MPa (B) as a function of emulsion passes through the valve homogenizer. All measurements plotted were carried out in the presence of 1% SDS (deflocculating agent) to avoid DSD influenced by the bridging flocculation phenomena [14]. When the homogenization pressure was 5 MPa (Figure 1A), unimodal DSD profiles were obtained in all cases; however, the distribution becomes narrower as emulsion passes increased. Thus, the distributions always started around 2 μm; however, the highest droplets moved from c.a. 40 to 20 μm, where the reduction of droplet sizes most important took place after passes 1 and 2. This reduction in droplet size can be expected since there is an increase in the energy input during emulsion processing as the number the passes increases [18]. Figure 1. Droplet size of W1/O/W2 multiple emulsions obtained after processing the secondary emulsion at 5 MPa (A) and 20 MPa (B), as a function emulsion passes through the high-pressure homogenizer. On the other hand, when the homogenization pressure increased up to 20 MPa (Figure 1B), the unimodal DSD was obtained only for one emulsion passing through the valve homogenizer. A higher number of passes involved the emergence of other tenuous droplet populations at the ends or the beginning of the main peak. Thus, for passes 2 and 3, shorter droplets were obtained due to the above-mentioned increase in energy input. However, this tendency changed for passes 4 and 5, where higher droplet populations were found. This behavior has been related to excessive processing energy, leading to a higher coalescence rate after passing the emulsion through the valve homogenizer [19]. In this sense, a comparison of Figure 1A,B show that an increase in pressure leads to a shift of the droplet size distribution profiles towards smaller sizes, although giving rise to apparently wider distributions. Moreover, an excess of energy (caused by the number of passes) led to an increase in droplet sizes due to coalescence. A similar influence of valve homogenizer pressure was found by Floury et al. [18] for conventional emulsions stabilized by whey protein isolate. In order to quantify the tendencies observed by droplet size distributions after processing the emulsions at two different homogenizer pressures (5 and 20 MPa) and passes (up to 5). Table 2 summarizes the Sauter mean diameter, the span parameter and the flocculation index (D3,2, span and FI, respectively) for all emulsions studied in this section. Figure 1. Droplet size of W 1 /O/W 2 multiple emulsions obtained after processing the secondary emulsion at 5 MPa ( A ) and 20 MPa ( B ), as a function emulsion passes through the high-pressure homogenizer. On the other hand, when the homogenization pressure increased up to 20 MPa (Figure 1B) , the unimodal DSD was obtained only for one emulsion passing through the valve homogenizer. A higher number of passes involved the emergence of other tenuous droplet populations at the ends or the beginning of the main peak. Thus, for passes 2 and 3, shorter droplets were obtained due to the above-mentioned increase in energy input. However, this tendency changed for passes 4 and 5, where higher droplet populations were found. This behavior has been related to excessive processing energy, leading to a higher coalescence rate after passing the emulsion through the valve homogenizer [ 19 ]. In this sense, a comparison of Figure 1A,B show that an increase in pressure leads to a shift of the droplet size distribution profiles towards smaller sizes, although giving rise to apparently wider distributions. Moreover, an excess of energy (caused by the number of passes) led to an increase in droplet sizes due to coalescence. A similar influence of valve homogenizer pressure was found by Floury et al. [ 18 ] for conventional emulsions stabilized by whey protein isolate. In order to quantify the tendencies observed by droplet size distributions after processing the emulsions at two different homogenizer pressures (5 and 20 MPa) and passes (up to 5). Table 2summarizes the Sauter mean diameter, the span parameter and the flocculation index (D 3,2 , span and FI, respectively) for all emulsions studied in this section. The values observed in this Table confirm the results previously observed in the drop size distribution curves since an increase in the number of steps led to a significant decrease in Span (higher droplet polydispersity). As for the D 3,2 , the increase in passes involved a decrease in this parameter, especially for the multiple emulsions processed at 5 MPa. However, more than three passes did not involve significant differences (p< 0.05). Foods 2022,11, 1367 8 of 17 Table 2. Parameters (D 3,2 , Span and FI) from droplet size distribution as a function of the number of emulsion passes through the high-pressure homogenizer and the two pressures studied (5 MPa and 20 MPa). Different superscript letters within a column indicate significant differences (p< 0.05). Passes 5 MPa 20 MPa D3,2 (µm) Span FI (%) D3,2 (µm) Span FI (%) 1 5.2 ±0.3 a2.2 ±0.1 a5.2 ±3.2 a4.1 ±0.3 a1.9 ±0.4 a20.0 ±8.0 a 24.7 ±0.2 b1.8 ±0.1 b9.5 ±2.1 b2.3 ±0.2 b2.2 ±0.3 a13.2 ±6.8 b 3 4.0 ±0.1 c1.6 ±0.2 c3.6 ±2.2 a1.9 ±0.2 b2.6 ±0.2 b6.4 ±4.2 c 4 4.3 ±0.2 c1.4 ±0.1 c14.0 ±4.1 c1.9 ±0.1 b2.2 ±0.3 a6.0 ±4.0 c 54.5 ±0.2 c,d 1.2 ±0.1 d9.0 ±2.9 b2.0 ±0.3 b4.0 ±0.2 c28.0 ±8.2 a The FI parameter accounts for the electrostatic interactions of oil droplets. Flocculation can modify the rheological behavior of emulsions, creating a network that prevents phase separation when highly concentrated emulsions are prepared. However, it can also promote droplet coalescence. In general, low flocculated emulsions were obtained. The standard deviation obtained in this parameter suggests that this flocculation was not too strong, and the system was deflocculated/flocculated by the simple mechanical agitation when the measurement was performed [20]. Thus, in terms of droplet size distribution, the input of higher energy to the system ( 20 MPa ) during emulsification processing led to emulsions with smaller droplet sizes (D 3,2 ). However, more monodispersed emulsions were achieved when the lowest pressure (5 MPa) was used. As for the number of passes, the optimal (emulsions more monodispersed and smaller droplet sizes) was found around 2–3 passes, regardless of the valve homogenizer pressure. The processing parameters selected were also based on the E E of the multiple emulsions generated. In fact, the objective of the present work is to maximize this key parameter. Thus, Table 3shows the E E for emulsions as a function of the number of emulsion passes through the high-pressure homogenizer and the two pressures studied (5 MPa and 20 MPa ). This table evidences a clear reduction in the E E when the homogenization pressure increases. In this sense, the encapsulation pressure was higher when emulsions were processed at 5 MPa than when they were processed at 20 MPa (i.e., 96.1 ± 1.8 vs. 87.3 ± 2.3 for three passes). Table 3. Encapsulation efficiency (E E ) for the emulsions as a function of the number of emulsion passes through the high-pressure homogenizer (steps) and the two pressures studied (5 MPa and 20 MPa). Different superscript letters within a column indicate significant differences (p< 0.05). Passes EE(%) 5 MPa EE(%) 20 MPa 1 97.0 ±2.1 a86.1 ±3.1 a 2 97.6 ±1.2 a54.1 ±6.1 b 3 96.1 ±1.8 a87.3 ±2.3 a 4 95.4 ±1.5 a74.3 ±5.6 c 5 97.0 ±1.1 a87.3 ±2.9 a No significant differences (p< 0.05) were found when the emulsions were processed at the lower pressure (5 MPa). On the other hand, when emulsions were processed at the highest pressure (20 MPa), a significant decrease in E E with the number of passes was found. However, this tendency was not clear for the emulsions processed at this pressure. Schuch et al. [ 21 ] evaluated the influence of processing conditions on the E E in double emulsions. They found that the E E directly depended on the size of W 1 /O (primary emulsions) since the bigger the primary emulsion droplets, the higher the encapsulation efficiency. However, an excess of energy in secondary emulsions led to a decrease in this parameter. Foods 2022,11, 1367 9 of 17 From these results, and their comparison with the results obtained from droplet size distributions, hereinafter, the emulsion will be processed at 5 MPa and three passes through the high-pressure homogenizer. These conditions generated emulsions with a high encapsulation efficiency, at the same time as the droplet size profiles obtained were unimodal, which has been related to higher emulsion stability than polydispersed droplet size distributions [22]. 3.3. Processing Conditions Optimization, Influence of PGPR Droplet Size Distribution Measurements The optimization of PGPR concentration in double emulsions was carried out using emulsions containing 25/75 of the (W 1 /O)/W 2 ratio. Moreover, according to the previous results obtained, the pressure in the valve homogenizer was 5 MPa, and emulsions were passed through it three times. Figure 2shows the droplet size distribution of W 1 /O/W 2 multiple emulsions obtained after using 1, 3 and 5 wt.% PGPR for the primary emulsion. All measurements were carried out in the presence of 1% SDS. The DSD profiles reveal practically single-mode curves, exhibiting a maximum value around 5 µ m. In the case of 3% and 5% of PGPR, the curves obtained were practically the same. However, the system containing 1% of PGPR showed a widening in the 1 µm range, resulting in a greater droplet dispersion. Foods 2022, 11, x FOR PEER REVIEW 9 of 18 found. However, this tendency was not clear for the emulsions processed at this pressure. Schuch et al. [21] evaluated the influence of processing conditions on the EE in double emulsions. They found that the EE directly depended on the size of W1/O (primary emulsions) since the bigger the primary emulsion droplets, the higher the encapsulation efficiency. However, an excess of energy in secondary emulsions led to a decrease in this parameter. From these results, and their comparison with the results obtained from droplet size distributions, hereinafter, the emulsion will be processed at 5 MPa and three passes through the high-pressure homogenizer. These conditions generated emulsions with a high encapsulation efficiency, at the same time as the droplet size profiles obtained were unimodal, which has been related to higher emulsion stability than polydispersed droplet size distributions [22]. 3.3. Processing Conditions Optimization, Influence of PGPR Droplet Size Distribution Measurements The optimization of PGPR concentration in double emulsions was carried out using emulsions containing 25/75 of the (W1/O)/W2 ratio. Moreover, according to the previous results obtained, the pressure in the valve homogenizer was 5 MPa, and emulsions were passed through it three times. Figure 2 shows the droplet size distribution of W1/O/W2 multiple emulsions obtained after using 1, 3 and 5 wt.% PGPR for the primary emulsion. All measurements were carried out in the presence of 1% SDS. The DSD profiles reveal practically single-mode curves, exhibiting a maximum value around 5 μm. In the case of 3% and 5% of PGPR, the curves obtained were practically the same. However, the system containing 1% of PGPR showed a widening in the 1 μm range, resulting in a greater droplet dispersion. 0.1 1 10 100 1000 0 2 4 6 8 10 12 Volume (%) Droplet size ( m m ) 1 wt. % PGPR 3 wt. % PGPR 5 wt. % PGPR Figure 2. Droplet size distribution of W1/O/W2 multiple emulsions obtained after using 1, 3 and 5 wt.% PGPR for the primary emulsion. Narrower distributions are desired in emulsions since it determines the potential destabilization processes (such as coalescence and further creaming) [23,24]. This change in DSD profile was also obtained by Altuntas [25] for double emulsions stabilized by PGPR– lecithin mixtures, which evidences the importance of PGPR in double emulsions. Table 4 summarizes parameters from droplet size distribution (D3,2, Span and FI) as well as EE to evaluate the influence of PGPR in these emulsions. This table indicates that the lowest D3,2 was obtained for the system containing 1 wt.% PGPR. However, this system also led to the highest Span value (higher polydispersity). Eisinaite et al. [7] found Figure 2. Droplet size distribution of W 1 /O/W 2 multiple emulsions obtained after using 1, 3 and 5 wt.% PGPR for the primary emulsion. Narrower distributions are desired in emulsions since it determines the potential destabilization processes (such as coalescence and further creaming) [ 23 , 24 ]. This change in DSD profile was also obtained by Altuntas [ 25 ] for double emulsions stabilized by PGPR–lecithin mixtures, which evidences the importance of PGPR in double emulsions. Table 4summarizes parameters from droplet size distribution (D 3,2 , Span and FI) as well as E E to evaluate the influence of PGPR in these emulsions. This table indicates that the lowest D 3,2 was obtained for the system containing 1 wt.% PGPR. However, this system also led to the highest Span value (higher polydispersity). Eisinaite et al. [ 7 ] found similar results; they observed that the initial droplet size of the double emulsions first increased with the increase in the amount of PGPR and then decreased. They suggest that this behavior could be a result of a combined effect: at higher concentrations of PGPR, the water transfer capacity was higher and swelling was promoted, while the interfacial tension of the droplets was lower, which leads to less swelling. Foods 2022,11, 1367 16 of 17 Although microstructural properties of emulsions can be modulated by the ratio between the primary and secondary emulsions ((W 1 /O)/W 2 ratio), all the emulsions generated without XG presented similar stability and EE (~95%). This result suggests that different products exhibiting different textures (i.e., mayonnaise, salad dressings, milkshake, etc.) could be developed based on these emulsions. However, emulsions without XG resulted in a higher destabilization over storage time. These results indicate that the formulation of these W1/O/W2 double emulsion systems allows the optimal encapsulation of the marker. Thus, the development of food matrices, in the form of multiple emulsions, would allow the encapsulation of bioactive compounds, which could result in the development of novelty food products. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11091367/s1, Table S1. Correlation of systems analyzed in this research, evaluating two parameters: the concentration of the oil phase ratio in the secondary emulsion as well as the effect of the thickening agent. Figure S1. Images of double emulsion droplets (emulsion containing 25/75 of (W1/O)/W2) obtained from optical microscopy. Author Contributions: Conceptualization, M.F., A.G. and C.C.-S.; methodology, C.C.-S.; software, M.F. and C.C.-S.; validation, C.C.-S.; formal analysis, M.F., A.G. and C.C.-S.; investigation, M.F.; resources, A.G. and C.C.-S.; data curation, M.F. and C.C.-S.; writing—original draft preparation, M.F.; writing—review and editing, C.C.-S. and A.G.; visualization, M.F.; supervision, C.C.-S. and A.G.; project administration, A.G.; funding acquisition, A.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Consejería de Transformación económica, Industria, Conocimiento y Universidades de la Junta de Andalucía-Agencia Andaluza del Conocimiento by the grant Ref. PY20_01046, Junta de Andalucía/FEDER, UE. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The datasets generated for this study are available on request to the corresponding author. Acknowledgments: The authors acknowledge Cristina Caro for her support in the lab. Authors also acknowledge the financial support of the Consejería de Transformación económica, Industria, Conocimiento y Universidades de la Junta de Andalucía-Agencia Andaluza del Conocimiento by the grant Ref. PY20_01046, Junta de Andalucía/FEDER, UE. Conflicts of Interest: The authors declare no conflict of interest. References 1. Araiza-Calahorra, A.; Akhtar, M.; Sarkar, A. Recent advances in emulsion-based delivery approaches for curcumin: From encapsulation to bioaccessibility. Trends Food Sci. Technol. 2018,71, 155–169. [CrossRef] 2. McClements, D.J. Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems: A review. Adv. Colloid Interface Sci. 2018,253, 1–22. [CrossRef] [PubMed] 3. Fathi, M.; Mozafari, M.R.; Mohebbi, M. Nanoencapsulation of food ingredients using lipid based delivery systems. Trends Food Sci. Technol. 2012,23, 13–27. 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