Concentrated soybean lecithin was characterized in terms of acetone-insoluble matter, hexane-insoluble matter, humidity (moisture content) and acid value according to the AOCS official methods (Ja 4–46, Ja 3–87, Ja 2b–87, Ja 6–55) (AOCS, 2001). Phospholipid composition was determined using HPLC-ELSD (Model: Agilent 1100, Agilent, Beijing, China) according to Becart et al., (1990). HPLC (Agilent 1100) equipped with a silica gel column (Lichrospher Si 60, 5 µm, 12 cm x 4 mm, Merck) and evaporative light scattering detector (ELSD, Alltech 3300) was used. Eluent A was chloroform/methanol/ammonium hydroxide, 80/19.5/0.5 (v/v/v) and eluent B was chloroform/methanol/water/ammonium hydroxide, 60/34/5.5/0.5 (v/v/v/v). The gradient was: 0–14 min: linear from A/B, 50/50 to 100% B, 14–25 min: hold 100% B, 25–30 min: 100% B to A/B, 50/50 and 15 min at A/B, 50/50 for column regeneration. The flow rate of the eluent was 1 mL/min. The pressure of the nebulizer gas (air) at ELSD was maintained at 3.2 bars and the drift tube temperature was set at 40 °C. The column temperature was also set at 40 °C. Samples (10 mg) were dissolved in 10 mL chloroform/methanol/water (70/25/5). 20 µL of the sample were injected into the HPLC. The sample (10 mg) was dissolved in (10 mL) chloroform-methanol (2:1) solution. 20 µL of the sample were injected into the HPLC. Analyses were performed in triplicate. The retention times of phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylethanolamine (Lyso PE), phosphatidylcholine (PC), lysophosphatidylcholine (Lyso PC) and phosphatidic acid (PA) were approximately 5.0, 7.0, 8.0, 8.8, 9.5 and 14.5 min, respectively. The percentages of individual constituents were also confirmed by quantitative column chromatography. Lecithin fatty acid composition was determined by gas chromatography (Model: Agilent HP6890, Agilent, Beijing, China) according to the AOCS Ce 2–66 method (AOCS, 2001). Analyses were performed in duplicate.

De-oiled lecithin was prepared from concentrated soybean lecithin through acetone- fractionation. Concentrated soybean lecithin was repeatedly extracted with cold acetone at 0 °C (acetone/gum ratio of 5:1 v/w; stirred for 30 min at 400 rpm for 3 times). The acetone solution fractions were subjected to centrifugation. After decantation of the acetone soluble part, the insoluble matter (PLs) located at the bottom of the centrifugation tube was collected and solvent traces were evaporated under vacuum conditions.

Concentrated soybean lecithin (50 g) and water (100 mL) were heated to 30 and 50 °C, respectively. They were then mixed and magnetically stirred for a period of 15 mins (with 400 rpm speed). The reaction was initiated by the addition of PLA₁ enzyme solution (0.5%; w/w of lecithin). The reaction was conducted at 50 °C for 25 min. All the reactions were carried out in duplicate. Aliquots (4 mL) were withdrawn from the reaction mixture at regular intervals for analysis of acid value according to the AOCS official method (Ja 6–55) (AOCS, 2001). At the end of the reaction, the reaction mixtures were heated at 110 °C for 30 min to inactivate the enzyme. Subsequently, the water was evaporated from the reaction mixture at 80 °C for 1 h.

Concentrated soybean lecithin (50 g) and solvents (100 mL tert-butanol/hexane) were heated at 50 and 60 °C, respectively. They were then mixed with stirring at 400 rpm with a magnetic bar. The reaction was initiated by the addition of PLA₁enzyme solution (0.5 %; w/w of lecithin). The reaction was conducted at 60 °C for about 6 h. All the reactions were carried out in duplicate. Aliquots (4 mL) were withdrawn from the stirred reaction mixture at regular intervals for analysis of acid value according to the AOCS official method (Ja 6–55) (AOCS, 2001). At the end of the reaction, the reaction mixtures were heated at 110 °C for 30 min to inactivate the enzyme. After that, the solvent was evaporated from the reaction mixture at 60 °C for 1 h.

Concentrated soybean lecithin (50 g) and glycerol (25 g) at a molar ratio of 1:4.5, tert-butanol (75g, 100 wt % of total substrates) and water (7.5 g, 10 wt % of total substrates) were mixed and mechanically stirred (500 rpm) at 50 °C. The reaction was initiated by adding a CAL B solution (5 wt% of lecithin) and PLA₁solution (5 wt% of lecithin). The reaction mixture was incubated at 50 °C for 8 h. At the end of the reaction, the reaction mixture was heated at 110 °C for 30 min to deactivate the enzymes. Solvent was evaporated at 60 °C for 30 min to recover the product. The composition of the reaction product was quantified using HPLC analysis.

Concentrated soybean lecithin (50 g) and glycerol (25 g) at a molar ratio of 1:4.5, and water (300 wt% of total substrates) were mixed and mechanically stirred (500 rpm) at 50 °C. The reaction was initiated by adding a CAL B solution (10 wt% of lecithin) and PLA₁solution (10 wt% of lecithin). The reaction mixture was incubated for 1 h at 50 °C. At the end of the reaction, the reaction mixture was heated at 110 °C for 30 min to deactivate the enzymes. Water was removed through rotary evaporation at 80 °C for 1 h. Composition of the reaction product was quantified using HPLC analysis.

Quantitative analyses of the glycerolyzed and hydrolyzed products were carried out by HPLC (Agilent 1100) equipped with a silica gel column (Lichrospher Si 60, 5 µm, 12 cm x 4 mm, Merck) and evaporative light scattering detector (ELSD, Alltech 3300). Eluent A was chloroform/methanol/ammonium hydroxide, 80/19.5/0.5 (v/v/v) and eluent B was chloroform/methanol/water/ammonium hydroxide, 60/34/5.5/0.5 (v/v/v/v). The gradient was: 0–14 min: linear from A/B, 50/50 to 100% B, 14–25 min: hold 100% B, 25–30 min: 100% B to A/B, 50/50 and 15 min at A/B, 50/50 for column regeneration. The flow rate of the eluent was 1 mL/min. The pressure of the nebulizer gas (air) in ELSD was maintained at 3.2 bars and the drift tube temperature was set at 40 °C. The column temperature was also set at 40 °C. The samples (10 mg) were dissolved in 10 mL chloroform/methanol/water (70/25/5). 20 µL of the sample were injected into the HPLC.

The HLB value was determined according to a method by Gupta et al., (1983) with slight modification. HLB standards (5.0, 5.2, 5.4, 5.6, 5.8, and 6.0) were prepared using cottonseed oil and turpentine. To measure the HLB values of lysolecithins, sample solutions were prepared (lysolecithins: standards: water [1:3:16] w/v/v). These sample solutions were mixed for 5 min at 200 rpm/min. 20 mL of the mixed solution were transferred to a graduated cylinder. Water separation from the mixtures was monitored for a period of 12 h. The equation used for the determination of HLB value was:

Where X=weight (mg) of high HLB value containing surfactant (Tween 80, turpentine), M₀=weight (mg) of standard oil required for the experiment, Y=low HLB value containing surfactant (span 80, cotton seed oil), HLB₀=HLB of sample (concentrated lecithin or related product), HLB₁=required HLB of cotton seed oil, HLB₂=required HLB of turpentine.

The calcium ion tolerance of PL and Lyso PL was in the following ascending order: concentrated lecithin < powder lecithin < lysolecithin from crude gum solution ≈ lysolecithin from concentrated lecithin < lysolecithin powder from lysolecithin (Figure 1A). The concentrated lecithin and its powder exhibited poor performance (at all the studied concentrations) in high calcium ion solution due to their higher sensitivity towards calcium ions. Lysolecithin has better emulsifying properties than concentrated lecithin-based emulsion indicating better calcium tolerance towards calcium ions. This is in agreement with previous findings that showed Lyso PE-stabilized emulsion did not flocculate in the presence of calcium ions and milk protein (Duin et al., 1963; Hoof et al., 2005).

At highly acidic conditions (pH=2), the ascending order of emulsion stability was as follows: concentrated lecithin < powder lecithin < lysolecithin from crude gum solution ≈ lysolecithin from concentrated lecithin < lysolecithin powder from lysolecithin (Figure 1B). Apart from lysolecithins prepared from crude gum solution and the concentrated lecithin, the powdered lysolecithin also had good emulsifying properties at pH 2 (strongly acidic condition). Whereas, when the concentrated lecithin, powdered lecithins were used as emulsifiers the oil phase in the emulsion was separated at a 24 h time period which led to complete demulsification of the emulsion. This finding is in agreement with previous observations that lysolecithin improves the stability of emulsion with high salt and over a wide range of pH. Therefore, it is used as an ingredient in foods requiring longer shelf life (Aoi, 1990).

At moderate acidity condition (pH=4) (Fig 1C), the ascending order of emulsion stability observed was as follows: concentrated lecithin ≈ powder lecithin < lysolecithin from crude gum solution ≈ lysolecithin from concentrated lecithin < lysolecithin powder from lysolecithin. At the moderate to highly acidic conditions (pH < 5) the lysolecithin and its powder exhibited good performance in stabilizing the emulsification system and the performance was much better than concentrated lecithin and its powder.

The emulsifying properties of concentrated and de-oiled lecithins are shown in Figure 1D. Powdered lysolecithin exhibited superior emulsifying properties. This is followed by lysolecithin from concentrated lecithin, lysolecithin from crude gum solution > powder lecithin > concentrated lecithin. It was observed that lysolecithin solutions were stable at least for 12 days. As lysolecithin exhibits high moisture retention, its solubilizing property, its lipophilic and hydrophilic moiety’s holding nature and emulsifying power are excellent. Especially, enzymatic hydrolyzed lecithin possesses technological and commercial advantages over native lecithins such as enhanced O/W emulsifying property, increased emulsion stability under acidic conditions and in the coexistence with salts, improved capability to bind proteins and starch and excellent mold- or pan-releasing property (Hirai et al., 1998; Erickson, 2008). Consequently, the demand for lysolecithins has increased in recent years.

The less hydrolyzed lecithins (lower AV and HLB) were ignored for the evaluation in the present study as their properties are expected to be more or less similar to normal concentrated lecithin. In general, lecithins that exhibit HLB values between 4–6, 7–9 and 8–10 are considered as water in oil (w/o) emulsifiers, wetting agents and oil in water (o/w) emulsifiers, respectively. In our case standard concentrated lecithins, lecithins with minor hydrolysis, lysolecithin 2 falls in the range of 4–6 HLB values and therefore, they are expected to have applications in w/o emulsions such as margarine, spreads, icings, frostings and petroleum emulsions. On the other hand, the optimally hydrolyzed lecithin such as lysolecithin 1 falls in the range of 8–10 HLB values and therefore, they will have the application in o/w emulsions such as mayonnaise, infant formulas, and hand and body lotions. Since the glycerolyzed lecithin also exhibited HLB near to 7.0, this product can be suitable in o/w emulsions and it will not be suitable for w/o emulsions.