Emulsifying Properties of Hydrolized Sunflower Lecithins by Phospholipases A2 of Different Sources

D. M. Cabezas1, R. Madoery2, B. W. K. Diehl3 and M. C. Tomás1 1Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA) – CCT La Plata – CONICET Facultad de Ciencias Exactas (FCE), Universidad Nacional de La Plata (FCE UNLP), La Plata 2Cátedra de Química Orgánica – Facultad de Ciencias Agrarias, Universidad Nacional de Córdoba (FCA UNC), Córdoba 3Spectral Service GmbH Laboratorium für Auftragsanalytik, Cologne 1,2Argentina 3Germany


Introduction
Lecithins are a mixture of acetone insoluble phospholipids, containing mainly phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), minor compounds such as phosphatidic acid (PA), and other minor substances such as carbohydrates and triglycerides (Schneider, 1989).The production of sunflower oil, in Argentina, is of utmost importance from an economic point of view (Franco, 2008).In this country, sunflower lecithin might represent an alternative to soybean lecithin because it is considered a non-GMO product, which is in accordance with the preference of some consumers.The introduction of changes in the original concentration of these phospholipids, by chemical or enzymatic modification of their structure can lead to obtain lecithins with different physicochemical and functional properties, with respect to native lecithin (van Nieuwenhuyzen & Tomás, 2008).The modification processes usually applied on native lecithins are the fractionation with ethanol (Sosada, 1993;Wu & Wang, 2004;Cabezas et al., 2009aCabezas et al., , 2009b) ) and the enzymatic hydrolysis (Schmitt & Heirman, 2007;Cabezas et al., 2011a).Native and modified lecithins are used in a wide range of industrial applications: nutritional, pharmaceutical applications, food, cosmetics, etc. (Prosise, 1985;Wendel, 2000).In the food industry, lecithin represents a multifunctional additive in the manufacture of chocolate, bakery and instant products, margarines, and mayonnaise, due to the characteristics of its phospholipids (van Nieuwenhuyzen, 1981).In particular, enzymatic hydrolyzed lecithin may present technological and commercial advantages over native lecithins: (1) enhanced O/W emulsifying property; (2) increased emulsion stability under acid conditions and in the coexistence with salts; (3) improved capability to bind proteins and starch; (4) excellent mold-or pan-releasing property.Consequently, the demand for lysolecithins was increasing in recent years (Hirai et al., 1998;Erickson, 2008).The main application of lecithin at the food industry is associated with its rol as emulsifier agent for dispersions or emulsions (Hernández & Quezada, 2008).Emulsions are thermodynamically unstable systems from a physicochemical point of view.In virtue of that, it is important to characterize and know their behaviour against different destabilization processes (flocculation, coalescence, creaming, etc.) (McClements, 1999).Enzymatic hydrolysis is carried out mainly by two groups of enzymes: phospholipases and lipases (Mustranta et al., 1995).Phospholipases A 2 catalyze the hydrolysis of the ester bond in the sn-2 position of glycerophospholipids, producing free fatty acids and the corresponding lysophospholipid.Advances in biotechnology and certain requirements of consumers (kosher or halal foods) have influenced the development of the production of microbial enzymes (bacteria, fungi, yeasts) which could be substitute of the traditionally obtained from porcine pancreas (Minchiotti, 2006;Cabezas et al., 2011b).The aim of this work was analyze the emulsifying activity of sunflower lysolecithins obtained by phospholipases A 2 from diverse sources: bacterial (LysoMax PLA 2 , Danisco) and porcine pancreas (Lecitase 10L, Novo Nordisk) in O/W systems.This study seeks to contribute to the oil industry with useful information for rescaling of the mentioned hydrolysis process, with the aim of increasing the aggregated value of sunflower lecithins.

Enzymatic hydrolysis process
Enzymatic hydrolysis was carried out in a thermostated reactor at laboratory scale, using 27 g of native sunflower lecithin and 18 ml of 0.4 M CaCl 2 .Initial pH was adjusted to 7 or 9 by adding 4 N NaOH solution.Then, the resulting mixture was set to the optimal temperature of each phospholipase, i.e. 60 ºC for porcine pancreatic PLA 2 and 50 ºC for microbial PLA 2 , which were incorporated in a concentration of 2.0% ml lipase per 100 g lecithin.Next, continuous agitation (50 rpm) was applied during 5 h.Evolution of hydrolysis process was followed by measuring pH, using a pH meter for solid samples (840049 Puncture Tip, Saen S.R.L.).Products of enzymatic hydrolysis were subjected to a sudden decrease in temperature to stop the process of hydrolysis and then deoiled using acetone, according to AOCS Official Method Ja 4-46, procedures 1-5 (Cabezas et al., 2011a).After that, samples were stored at 0 °C.The hydrolysis process was carried out in duplicate.
Also, native sunflower lecithin was deoiled with acetone obtaining the deoiled sunflower lecithin (DSL).DSL was used as a sample control.Deoiling procedure was performed by duplicate.

Phospholipid composition 2.3.1 Sample preparation
100 mg of each hydrolyzed sample were diluted in 1 ml of deuterated chloroform, 1 ml of methanol and 1 ml of Cs-EDTA (pH 8).The organic layer was separated after 15 min shaking, and analyzed by 31 P NMR (Cabezas et al., 2009a).

Quantitative
31 P NMR analysis Quantitative 31 P NMR analysis was carried out in a Bruker Avance 600 MHz automatic spectrometer using triphenyl phosphate as internal standard (Spectral Service GmbH, Köln, Germany) (Diehl, 1997;2001;2008).Phospholipid content of samples obtained under different conditions of enzymatic hydrolysis, was determined by this spectroscopic technique.

Oil-in-water (O/W) emulsions preparation
Commercial sunflower oil was used to prepare oil-in-water (O/W) emulsions with a formulation of 30:70 (wt/wt) according to Pan et al., 2004.Emulsions were prepared at room temperature in an Ultra-Turrax T25 homogenizer using S 25 N-10 G dispersing tool (7.5 mm rotor diameter) at 10,000 rpm for 1 min, with the addition of the different modified sunflower lecithins in a range of 0.1-2.0%(wt/wt).This process was carried out in triplicate for each case.

Optical characterization of emulsions
The backscattering of light was measured using a QuickScan Vertical Scan Analyzer (Coulter Corp., Miami, FL).The backscattering of monochromatic light (λ = 850 nm) of the emulsions was determined as a function of the height of the sample tube (ca.65 mm) in order to quantify the rate of the different destabilization processes during the first 90 min.This methodology allowed to discriminate between particle migration (sedimentation, creaming) and particle size variation (flocculation, coalescence) processes (Pan et al., 2002).The basis of the vertical scan analyzer profiles has been exhaustively studied by Mengual (Mengual et al., 1999).

Particle size measurements
Particle size distribution, and De Brouckere (D[4,3]) and Sauter (D[3,2]) mean diameters of particles of the emulsions were determined with a particle size analyzer (Malvern Mastersizer 2000E, Malvern Instruments Ltd., Worcestershire,U.K.).Samples were diluted in the water bath of the dispersion system (Hydro 2000MU), which is a laser diffraction based particle size analyzer (Márquez & Wagner, 2010).This determination was carried out in triplicate for each case.

Statistical analysis
Data were evaluated by analysis of variance (ANOVA) using the software Systat ® 12.0 (Systat, 2007).For this purpose, differences were considered significant at p < 0.05.

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 A 2 , 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 PLA 2 (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 PLA 2 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 31 P NMR equipment when the pancreatic porcine PLA 2 was used.

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.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).[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 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.

Conclusions
The best emulsifying properties exhibited by hydrolyzed sunflower lecithins (LHM7, LHP7, LHM9, LHP9) in comparison with the native lecithin could indicate that their application by the local oil industry could revalue this by-product of the refining process of crude sunflower oil.The use of a microbial phospholipase A 2 gives the possibility to obtain diverse sunflower lysolecithins, which functionality could be applied to the development of foods with kosher and halal certification.

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