**1. Introduction**

38 Food Industrial Processes – Methods and Equipment

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84569 414 2, Boca Raton, Florida

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., 2009a, 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

Emulsifying Properties of Hydrolized

**2.3 Phospholipid composition 2.3.1 Sample preparation** 

**2.3.2 Quantitative 31P NMR analysis** 

**2.4 Oil-in-water (O/W) emulsions preparation** 

**2.5 Optical characterization of emulsions** 

shaking, and analyzed by 31P NMR (Cabezas et al., 2009a).

duplicate.

technique.

for each case.

(Mengual et al., 1999).

triplicate for each case.

**2.7 Statistical analysis** 

**2.6 Particle size measurements** 

Sunflower Lecithins by Phospholipases A2 of Different Sources 41

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

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

Quantitative 31P 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

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

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

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

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.

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 A2 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 A2 from diverse sources: bacterial (LysoMax PLA2, 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.

### **2. Materials and methods**

#### **2.1 Materials**

Native sunflower lecithin was used as starting material, and was provided by a local oil industry (Vicentin S.A.I.C.). Enzymatic hydrolysis processes were carried out using a porcine pancreatic PLA2 (Lecitase 10L, Novo Nordisk) and a microbial PLA2 (*Streptomyces violaceoruber*, LysoMax PLA2, Danisco). All solvents used were of analytical grade.

The sunflower lecithin used as starting material presented the following composition: 43.1% phospholipids (16.5% PI, 16.2% PC, 5.3% PE, and 5.1% minor phospholipids), 33.4% oil, and 23.5% of other compounds (glycolipids, complex carbohydrates).

#### **2.2 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 CaCl2. 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 PLA2 and 50 ºC for microbial PLA2, 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.

### **2.3 Phospholipid composition**

#### **2.3.1 Sample preparation**

40 Food Industrial Processes – Methods and Equipment

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;

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 A2 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

The aim of this work was analyze the emulsifying activity of sunflower lysolecithins obtained by phospholipases A2 from diverse sources: bacterial (LysoMax PLA2, 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.

Native sunflower lecithin was used as starting material, and was provided by a local oil industry (Vicentin S.A.I.C.). Enzymatic hydrolysis processes were carried out using a porcine pancreatic PLA2 (Lecitase 10L, Novo Nordisk) and a microbial PLA2 (*Streptomyces* 

The sunflower lecithin used as starting material presented the following composition: 43.1% phospholipids (16.5% PI, 16.2% PC, 5.3% PE, and 5.1% minor phospholipids), 33.4% oil, and

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 CaCl2. 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 PLA2 and 50 ºC for microbial PLA2, 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

*violaceoruber*, LysoMax PLA2, Danisco). All solvents used were of analytical grade.

23.5% of other compounds (glycolipids, complex carbohydrates).

obtained from porcine pancreas (Minchiotti, 2006; Cabezas et al., 2011b).

Erickson, 2008).

**2. Materials and methods** 

**2.2 Enzymatic hydrolysis process** 

**2.1 Materials** 

duplicate.

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 31P NMR (Cabezas et al., 2009a).
