**3.1 Compositional analysis of modified lecithins**

The phospholipid composition of different modified sunflower lecithins obtained in this chapter is shown in Table 1. The sunflower hydrolyzed lecithins with a porcine pancreatic (SHLP) and microbial (SHLM) phospholipase A2, at different levels of pH (7, 9), recorded a marked difference regarding the phospholipid composition in relation to the native sunflower lecithin (DSL). The hydrolyzed lecithins presented a high concentration of major lysophospholipids (> 64.9 mol LPL/ mol of total PL) compared to the native sunflower lecithin (≈ 1.5%), showing the efficiency of the enzymatic hydrolysis processes. In particular, the pancreatic PLA2 (SHLP7, SHLP9) produced a higher hydrolysis degree of the main phospholipids in comparison with the microbial phospholipase (SHLM7, SHLM9).

PC presented a high degree of hydrolysis in all performed conditions on the hydrolysis processes. These results can be correlated with the ones described by Penci, 2010. In that work, it was reported that phosphatidylcholine is the phospholipid with higher tendency to be hydrolyzed when using a porcine pancreatic PLA2 with a very low amount of sunflower lecithins (1 mg). In this way, the residual PC and PI concentration in the hydrolyzed lecithin was lower than the detection limit of the 31P NMR equipment when the pancreatic porcine PLA2 was used.

#### **3.2 Optical characterization of O/W emulsions**

Stability of the different O/W emulsions (30:70 wt/wt) was studied recording the backscattering (BS) profiles as a function of the cell length and time, by a vertical scan analyzer (QuickScan). For instance, Figure 1 shows two typical profiles obtained for emulsions with the addition of 0.1% of DSL and SHLP9.

**PL / 100g lecithin** 62.8 40.6 45.9 38.7 44.4

The phospholipid composition of different modified sunflower lecithins obtained in this chapter is shown in Table 1. The sunflower hydrolyzed lecithins with a porcine pancreatic (SHLP) and microbial (SHLM) phospholipase A2, at different levels of pH (7, 9), recorded a marked difference regarding the phospholipid composition in relation to the native sunflower lecithin (DSL). The hydrolyzed lecithins presented a high concentration of major lysophospholipids (> 64.9 mol LPL/ mol of total PL) compared to the native sunflower lecithin (≈ 1.5%), showing the efficiency of the enzymatic hydrolysis processes. In particular, the pancreatic PLA2 (SHLP7, SHLP9) produced a higher hydrolysis degree of the main phospholipids in comparison with the microbial phospholipase (SHLM7,

PC presented a high degree of hydrolysis in all performed conditions on the hydrolysis processes. These results can be correlated with the ones described by Penci, 2010. In that work, it was reported that phosphatidylcholine is the phospholipid with higher tendency to be hydrolyzed when using a porcine pancreatic PLA2 with a very low amount of sunflower lecithins (1 mg). In this way, the residual PC and PI concentration in the hydrolyzed lecithin was lower than the detection limit of the 31P NMR equipment when the pancreatic porcine

Stability of the different O/W emulsions (30:70 wt/wt) was studied recording the backscattering (BS) profiles as a function of the cell length and time, by a vertical scan analyzer (QuickScan). For instance, Figure 1 shows two typical profiles obtained for

Table 1. Phospholipid (PL) composition of modified sunflower lecithins by 31PNMRa

**3. Results and discussion** 

SHLM9).

PLA2 was used.

**3.1 Compositional analysis of modified lecithins** 

**3.2 Optical characterization of O/W emulsions** 

emulsions with the addition of 0.1% of DSL and SHLP9.

**PC** 36.7 7.4 6.7 < 0.1 < 0.1 **1-LPC** < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 **2-LPC** 1.5 39.2 39.4 46.1 45.5 **PI** 35.3 11.2 12.3 < 0.1 < 0.1 **LPI** < 0.1 18.6 15.5 26.5 27.9 **PE** 15.1 3.7 6.4 1.0 0.7 **LPE** < 0.1 8.8 9.0 10.7 13.0 **APE** 1.8 < 0.1 < 0.1 < 0.1 < 0.1 **PA** 5.2 3.1 3.4 2.5 2.0 **LPA** < 0.1 1.0 1.0 5.1 4.1 **Other** 4.5 7.1 6.4 8.0 6.7

**DSL SHLM9 SHLM7 SHLP9 SHLP7** 

a Mean values are shown (*n* = 3). The coefficient of variation was lower than 5%

Fig. 1. Backscattering (%BS) profiles of O/W emulsions (30:70 wt/wt) with the addition of: (a) DSL, 0.1%; (b) SHLP, 0.1%

Emulsifying Properties of Hydrolized

**3.3 Particle size distribution** 

comparison with DSL.

Sunflower Lecithins by Phospholipases A2 of Different Sources 45

Fig. 3. Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sunflower lecithins in Zone II (40-45 mm). Mean values (*n* = 3) ± sd

Particle size distribution in volume and surface of O/W emulsions obtained with different modified lecithins was measured just after emulsification (t=0); corresponding results can be seen in Figures 4 and 5, respectively. These distributions presented a bimodal or trimodal character depending on the concentration of aggregated lecithin, and the following particle size populations: (I) particle size < 4 m; (II) particle size between 4 and 30 m; (III) particle size > 30 m. In this sense, only SHLP7 showed a trimodal character for all concentrations assayed. It should be noted that the SHLM7 presented a bimodal character for concentration in the range 0.1-0.5%, but with a high percentage of particles of the population II in

In order to complete the analysis of particle size distribution, Figure 6 depicts the evolution of De Brouckere (D [4,3]) and Sauter (D [3,2]) mean diameters as a function of the concentration of the different emulsifiers. Hydrolyzed lecithins generated values of D [4,3] and D [3,2] significantly lower than those corresponding to DSL. These results are correlated with the high stability of the O/W emulsions recorded when using hydrolyzed lecithins, considering the main destabilization processes determined by the corresponding QuickScan profiles (creaming or coalescence). It is worth to note that a high concentration of small particles produces a slow

creaming process, according to the Stokes' law (McClements, 1999; Palazolo, 2006).

The creaming destabilization process (i.e. migration of oil particles to the upper portion of the tube) is evidenced by a decrease of %BS values at the bottom of the tube. The QuickScan profiles corresponding to the zone I (10-20 mm) showed an increase of the emulsion stability against the creaming process, as a function of increasing concentration of different modified lecithins (Fig. 2). In particular, the hydrolyzed lecithins (SHLP and SHLM) generated a high stability in O/W emulsions than DSL, over the studied range of concentration. Moreover, O/W emulsions with 0.1-0.5% of DSL showed a sharp decrease of %BS in the Zone I. The tube zone between 40-45 mm (Zone II) is characterized by the accumulation of oil

droplets after the creaming process (cream phase); Figure 3 shows the %BS values vs. time in Zone II. Emulsions formulated with hydrolyzed lecithins presented higher %BS values than those obtained using DSL, for all concentrations studied. The higher levels of %BS and the greater stability of these emulsions would be associated with the formation of dense cream phases with a lower proportion of continuous phase inside (Palazolo, 2006). However, emulsions with 0.1 -0.5% of DSL did not allow the formation of the cream phase. These results are related to the rapid decrease of %BS and the formation of an oil layer in the upper part of the tube (Fig. 1a) suggesting the occurrence of a cream phase destabilization by coalescence (Pan et al., 2002).

Fig. 2. Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sunflower lecithins in Zone I (10-20 mm). Mean values (*n* = 3) ± sd

The creaming destabilization process (i.e. migration of oil particles to the upper portion of the tube) is evidenced by a decrease of %BS values at the bottom of the tube. The QuickScan profiles corresponding to the zone I (10-20 mm) showed an increase of the emulsion stability against the creaming process, as a function of increasing concentration of different modified lecithins (Fig. 2). In particular, the hydrolyzed lecithins (SHLP and SHLM) generated a high stability in O/W emulsions than DSL, over the studied range of concentration. Moreover,

The tube zone between 40-45 mm (Zone II) is characterized by the accumulation of oil droplets after the creaming process (cream phase); Figure 3 shows the %BS values vs. time in Zone II. Emulsions formulated with hydrolyzed lecithins presented higher %BS values than those obtained using DSL, for all concentrations studied. The higher levels of %BS and the greater stability of these emulsions would be associated with the formation of dense cream phases with a lower proportion of continuous phase inside (Palazolo, 2006). However, emulsions with 0.1 -0.5% of DSL did not allow the formation of the cream phase. These results are related to the rapid decrease of %BS and the formation of an oil layer in the upper part of the tube (Fig. 1a) suggesting the occurrence of a cream phase destabilization

Fig. 2. Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sunflower lecithins in Zone I (10-20 mm). Mean values (*n* = 3) ± sd

O/W emulsions with 0.1-0.5% of DSL showed a sharp decrease of %BS in the Zone I.

by coalescence (Pan et al., 2002).

Fig. 3. Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sunflower lecithins in Zone II (40-45 mm). Mean values (*n* = 3) ± sd

#### **3.3 Particle size distribution**

Particle size distribution in volume and surface of O/W emulsions obtained with different modified lecithins was measured just after emulsification (t=0); corresponding results can be seen in Figures 4 and 5, respectively. These distributions presented a bimodal or trimodal character depending on the concentration of aggregated lecithin, and the following particle size populations: (I) particle size < 4 m; (II) particle size between 4 and 30 m; (III) particle size > 30 m. In this sense, only SHLP7 showed a trimodal character for all concentrations assayed. It should be noted that the SHLM7 presented a bimodal character for concentration in the range 0.1-0.5%, but with a high percentage of particles of the population II in comparison with DSL.

In order to complete the analysis of particle size distribution, Figure 6 depicts the evolution of De Brouckere (D [4,3]) and Sauter (D [3,2]) mean diameters as a function of the concentration of the different emulsifiers. Hydrolyzed lecithins generated values of D [4,3] and D [3,2] significantly lower than those corresponding to DSL. These results are correlated with the high stability of the O/W emulsions recorded when using hydrolyzed lecithins, considering the main destabilization processes determined by the corresponding QuickScan profiles (creaming or coalescence). It is worth to note that a high concentration of small particles produces a slow creaming process, according to the Stokes' law (McClements, 1999; Palazolo, 2006).

Emulsifying Properties of Hydrolized

Sunflower Lecithins by Phospholipases A2 of Different Sources 47

Fig. 5. Surface particle size distribution for O/W emulsions with the addition of different

Fig. 6. De Brouckere (D[4,3]) and Sauter (D[3,2]) mean diameters for O/W emulsions with

the addition of different modified sunflower lecithins. Mean values (n = 3) ± sd

modified sunflower lecithins. Mean values (n = 3)

Fig. 4. Volume particle size distribution for O/W emulsions with the addition of different modified sunflower lecithins. Mean values (n = 3)

The hydrophilic-lipophilic balance value (HLB) is often used in connection with the performance of emulsifiers (McClements, 1999). The high concentration of hydrophilic phospholipids (lysophospholipids) presented in the hydrolyzed lecithins (SHLP and SHLM) increase this empirical value. In this sense, according to Carlsson (Carlsson, 2008), these modified lecithins with higher HLB values presented best properties as O/W emulsifying agents.

Also, the phase structure at the interface of the different phospholipids influences the emulsion formation and stability (van Nieuwenhuyzen & Tomás, 2008). LPC and LPE form hexagonal wide spread clusters. These structures have a great importance for the stabilisation of O/W emulsions. This behaviour is in relation to the low mean diameters and the high concentration of small particle populations recorded in emulsions using sunflower hydrolyzed lecithins (Figs. 4-6). However, PE gives reversed hexagonal phase, which are more difficult to arrange at the interface (van Nieuwenhuyzen, 1998). The presence of PE could explain the minor characteristics as emulsifying agent of DSL, and the high mean diameters when was using SHLM in contrast to when using SHLP (Fig. 6).

Taking into account the results presented in Figures 2 to 6, the addition of a concentration between 0.5 and 1.0% of hydrolyzed lecithin (SHLP, SHLM) is enough for covering all droplets surface. High concentrations of this modified lecithin do not show significant differences in the % BS values, nor in the mean particle sizes. On the other hand, concentration levels higher than 0.1% of hydrolyzed lecithins from different phospholipases at different initial pH levels showed similar characteristics in terms of their emulsifying activity. However, DSL presented an improved in the stability of O/W emulsions as a function of increasing concentration.

Fig. 4. Volume particle size distribution for O/W emulsions with the addition of different

The hydrophilic-lipophilic balance value (HLB) is often used in connection with the performance of emulsifiers (McClements, 1999). The high concentration of hydrophilic phospholipids (lysophospholipids) presented in the hydrolyzed lecithins (SHLP and SHLM) increase this empirical value. In this sense, according to Carlsson (Carlsson, 2008), these modified lecithins with higher HLB values presented best properties as O/W emulsifying

Also, the phase structure at the interface of the different phospholipids influences the emulsion formation and stability (van Nieuwenhuyzen & Tomás, 2008). LPC and LPE form hexagonal wide spread clusters. These structures have a great importance for the stabilisation of O/W emulsions. This behaviour is in relation to the low mean diameters and the high concentration of small particle populations recorded in emulsions using sunflower hydrolyzed lecithins (Figs. 4-6). However, PE gives reversed hexagonal phase, which are more difficult to arrange at the interface (van Nieuwenhuyzen, 1998). The presence of PE could explain the minor characteristics as emulsifying agent of DSL, and the high mean

Taking into account the results presented in Figures 2 to 6, the addition of a concentration between 0.5 and 1.0% of hydrolyzed lecithin (SHLP, SHLM) is enough for covering all droplets surface. High concentrations of this modified lecithin do not show significant differences in the % BS values, nor in the mean particle sizes. On the other hand, concentration levels higher than 0.1% of hydrolyzed lecithins from different phospholipases at different initial pH levels showed similar characteristics in terms of their emulsifying activity. However, DSL presented an improved in the stability of O/W emulsions as a

diameters when was using SHLM in contrast to when using SHLP (Fig. 6).

modified sunflower lecithins. Mean values (n = 3)

function of increasing concentration.

agents.

Fig. 5. Surface particle size distribution for O/W emulsions with the addition of different modified sunflower lecithins. Mean values (n = 3)

Fig. 6. De Brouckere (D[4,3]) and Sauter (D[3,2]) mean diameters for O/W emulsions with the addition of different modified sunflower lecithins. Mean values (n = 3) ± sd

Emulsifying Properties of Hydrolized

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