**Antioxidant and Emulsifying Properties of Modified Sunflower Lecithin by Fractionation with Ethanol-Water Mixtures**

Dario M. Cabezas, Estefanía N. Guiotto, Bernd W. K. Diehl and Mabel C. Tomás

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53165

## **1. Introduction**

[235] Romero F.J., Garcıa L.A., Salas J.A., Dıaz M., Quiros L.M. Production, purification

[236] Ustariz F., Laca A., Garcıa L.A., Dıaz M. Mixed cultures of *Serratia marcescens* and

grown in whey. Process Biochemistry 2001;36 507–515

588 Food Industry

whey. J. of Applied Microbiology 2007;103 864–870.

and partial characterization of two extracellular proteases from *Serratia marcescens*

*Kluyveromyces fragilis* for simultaneous protease production and COD removal of

Lecithins are a mixture of acetone insoluble phospholipids, containing mainly phosphatidyl‐ choline (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA), and other minor substances such as carbohydrates and triglycerides [1-3]. The produc‐ tion of sunflower oil in Argentina, is of utmost importance from an economic point of view [4]. In this country, sunflower lecithin could represent an alternative to soybean lecithin be‐ cause it is considered a non-GMO product, which is in accordance with the preference of some consumers [5].

Lecithins, in native or modified state, are used in a wide range of industrial applications: di‐ etetic, pharmaceutical, food, cosmetics, etc. This by-product of the oil industry represents a multifunctional additive for the manufacture of chocolate, bakery and instant products, margarine, mayonnaise, due to the characteristics of its phospholipids [6-8].

The introduction of changes in the relative concentration of the original phospholipid com‐ position of lecithin can originate enriched fractions in certain phospholipids with different physicochemical and functional properties for diverse industrial purposes [9-11].

The fractionation process by ethanol or ethanol:water mixtures takes advantage of the dif‐ ferent solubility of the phospholipids in this solvent. PC is readily soluble in ethanol where‐ as PI and PA are virtually insoluble. Phosphatidylethanolamine can be found in both fractions. This process can be carried out alone or in combination with other techniques such

as chromatography as a further purification step, especially for pharmaceutical, cosmetic and dietetic industry [12-13].

(Figure 1). Then, both fractions were stored at 0 ºC. Fractionation and deoiling procedures

Antioxidant and Emulsifying Properties of Modified Sunflower Lecithin by Fractionation with Ethanol-Water Mixtures

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591

**Figure 1.** Flow diagram for deoiling and ethanol fractionation of native sunflower lecithin

**Figure 2.** Spectrum of a phospholipidic sample

were performed in duplicate.

The main application of lecithin at the food industry is associated with its rol as emulsifying agent for dispersions or emulsions [14]. Emulsions are thermodynamically unstable systems from a physicochemical point of view. In virtue of that, it is important to characterize their behaviour against different destabilization processes (flocculation, coalescence, creaming, etc.) [15]. PC enriched fraction, due to its high PC/PE ratio and the lamellar phase structure of the PC at the interface between oil in water, is recognized to be a good oil-in-water (O/W) emulsifier [12,16,17].

On the other hand, PLs can contribute to an improvement of the oxidative stability of fats and oils. Various antioxidative mechanisms have been proposed for the phospholipid ac‐ tions. For example, the amino functions of PC, PS, or PE, or the sugar moiety of PI have been shown to have metal-chelating properties and PC and PE presented a synergistic effect, with phenolic antioxidants such as tocopherols and flavonoids [18, 19].

The objective of this work was to evaluate the antioxidant and emulsifying properties of modified sunflower lecithins by fractionation with ethanol-water mixtures. In this sense, this study seeks to contribute to the food industry with useful information about developing tai‐ lor-made surface-active emulsifiers.

## **2. Materials and methods**

#### **2.1. Materials**

Native sunflower lecithin was provided by a local oil industry (Vicentin S.A.I.C.).. This leci‐ thin present a phospholipid composition of 43.1% (PC 16.2%, PI 16.5%, PE 5.3%, minor PLs 5.1%), 23.5% other compounds (glycolipids, complex carbohydrates), 33.4% oil.

Sunflower lecithin was deoiled with acetone, according to AOCS Official Method Ja 4-46, procedures 1–5 [20], obtaining the deoiled sunflower lecithin (DSL) (Figure 1). Then, DSL was stored at 0 ºC. This modified lecithin was used as control sample. Deoiling procedure was performed in duplicate.

#### **2.2. Sunflower lecithin fractionation**

Fractionation process was performed to 30 g of native sunflower lecithin with the addition of extraction solvents with different ethanol/water ratio (96:4, 100:0) using an ethanol/leci‐ thin ratio of 3:1. These samples were incubated in a water bath at 65 °C during 60 min with moderate agitation (60 rpm), and then centrifuged at 1880 g, 10 ºC during 10 min. After‐ wards, the corresponding ethanolic extracts were obtained and ethanol was eliminated by evaporation under vacuum [17].

Ethanol soluble phases were further deoiled with acetone, according to AOCS Official Meth‐ od Ja 4-46, procedures 1–5, obtaining the different PC enriched fractions (PCF 96, PCF 100) (Figure 1). Then, both fractions were stored at 0 ºC. Fractionation and deoiling procedures were performed in duplicate.

**Figure 1.** Flow diagram for deoiling and ethanol fractionation of native sunflower lecithin

**Figure 2.** Spectrum of a phospholipidic sample

as chromatography as a further purification step, especially for pharmaceutical, cosmetic

The main application of lecithin at the food industry is associated with its rol as emulsifying agent for dispersions or emulsions [14]. Emulsions are thermodynamically unstable systems from a physicochemical point of view. In virtue of that, it is important to characterize their behaviour against different destabilization processes (flocculation, coalescence, creaming, etc.) [15]. PC enriched fraction, due to its high PC/PE ratio and the lamellar phase structure of the PC at the interface between oil in water, is recognized to be a good oil-in-water (O/W)

On the other hand, PLs can contribute to an improvement of the oxidative stability of fats and oils. Various antioxidative mechanisms have been proposed for the phospholipid ac‐ tions. For example, the amino functions of PC, PS, or PE, or the sugar moiety of PI have been shown to have metal-chelating properties and PC and PE presented a synergistic effect, with

The objective of this work was to evaluate the antioxidant and emulsifying properties of modified sunflower lecithins by fractionation with ethanol-water mixtures. In this sense, this study seeks to contribute to the food industry with useful information about developing tai‐

Native sunflower lecithin was provided by a local oil industry (Vicentin S.A.I.C.).. This leci‐ thin present a phospholipid composition of 43.1% (PC 16.2%, PI 16.5%, PE 5.3%, minor PLs

Sunflower lecithin was deoiled with acetone, according to AOCS Official Method Ja 4-46, procedures 1–5 [20], obtaining the deoiled sunflower lecithin (DSL) (Figure 1). Then, DSL was stored at 0 ºC. This modified lecithin was used as control sample. Deoiling procedure

Fractionation process was performed to 30 g of native sunflower lecithin with the addition of extraction solvents with different ethanol/water ratio (96:4, 100:0) using an ethanol/leci‐ thin ratio of 3:1. These samples were incubated in a water bath at 65 °C during 60 min with moderate agitation (60 rpm), and then centrifuged at 1880 g, 10 ºC during 10 min. After‐ wards, the corresponding ethanolic extracts were obtained and ethanol was eliminated by

Ethanol soluble phases were further deoiled with acetone, according to AOCS Official Meth‐ od Ja 4-46, procedures 1–5, obtaining the different PC enriched fractions (PCF 96, PCF 100)

5.1%), 23.5% other compounds (glycolipids, complex carbohydrates), 33.4% oil.

phenolic antioxidants such as tocopherols and flavonoids [18, 19].

and dietetic industry [12-13].

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emulsifier [12,16,17].

lor-made surface-active emulsifiers.

**2. Materials and methods**

was performed in duplicate.

**2.2. Sunflower lecithin fractionation**

evaporation under vacuum [17].

**2.1. Materials**

## **2.3. Phospholipid composition**

Phospholipid composition of samples obtained after different modification processes was determined by 31P NMR analysis in a Bruker Avance 600 MHz automatic spectrometer, us‐ ing triphenyl phosphate as internal standard (Spectral Service GmbH, Köln, Germany) (Fig‐ ure 2) [21-23]. For this purpose, 100 mg of each modified lecithin were diluted in 1 mL of deuterated chloroform, 1 mL of methanol and 1 mL of 0.2 M Cs-EDTA (pH 8.0). The organic layer was separated after 15 min shaking, and analyzed by the described spectroscopic tech‐ nique. Phospholipid composition of the different modified sunflower lecithins (MSLs) was expressed in terms of molar concentration (mol / 100 mol lecithin) [17].

**2.7. Statistical analysis**

**3.1. Phospholipid composition**

**3.2. Rancimat analysis**

a

fractions analyzed by 31PNMR

shows ti

**3. Results**

Data were evaluated by analysis of variance (ANOVA) using the software Systat® 12.0 [29].

Antioxidant and Emulsifying Properties of Modified Sunflower Lecithin by Fractionation with Ethanol-Water Mixtures

The quantitative analysis of phospholipids was performed by 31PNMR which represents a modern and the most sophisticated methodology for evaluating the composition of lecithins, since it is possible to obtain a separate signal for each phospholipid class [9, 21]. The results pre‐ sented in Table 1, evidenced the high solubility of the phosphatidylcholine in the different ethanolic solvents assayed. 31PNMR determinations of the different enriched PC fractions (PCF 96 and PCF 100) exhibited an important concentration of PC (>71.0 %) as well as low values of

The oxidative stability of the refined sunflower oil (control) and the activity of different

sis. This methodology can be used to evaluate the efficiency of various synthetic or natural antioxidants to stabilize fats and oils against an accelerated oxidation test [30]. Figure 3

> **PL DSL a PCF 96 a PCF 100 a** PC 36.7 71.2 76.3 1-LPC < 0.1 < 0.1 < 0.1 2-LPC 1.5 3.3 2.8 PI 35.3 7.4 3.6 LPI < 0.1 < 0.1 < 0.1 PE 15.1 12.2 10.6 LPE < 0.1 0.8 0.4 PA 5.0 1.3 1.4 LPA < 0.1 < 0.1 < 0.1 Others PL 6.4 3.8 4.9

for the modified sunflower lecithins used at different concentrations.

Values represent means (n = 2). The coefficient of variation was lower than 6%

**Table 1.** Percentage phospholipid composition (mol PL / mol total PL) of deoiled sunflower lecithin and PC-enriched

) by a Rancimat analy‐

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593

PI (<8%) in comparison with the native and deoiled sunflower lecithin assayed.

modified lecithins were evaluated determining the induction time (ti

For this purpose, differences were considered significant at *p* <0.05.

### **2.4. Antioxidant properties**

The antioxidant properties of the MSLs were evaluated by the Rancimat (Mod 679, Met‐ rohm) method. 5 g of sunflower oil were added with different concentration of the analyzed samples (500-2000 ppm), heated at 98 °C, air flow 20 L/h. Stability was expressed as the in‐ duction time, according to Gutiérrez [24].

Also, the highest antioxidant concentration was selected for each modified lecithin. The same procedure was carried out with previous thermal treatments at 120 and 160 °C for 1 h, adding the modified lecithins before heating. Refined sunflower oil with and without previ‐ ous heat treatments were used as control samples.

Oil tocopherol content was determined by normal phase HPLC using a Hewlett Packard chromatography system (HPLC Hewlett Packard 1050 Series, Waldbronn, Germany) equip‐ ped with a fluorescence detector Agilent 1100 Series (Agilent Technology, Palo Alto, CA, USA) following the procedures described in IUPAC 2.432 [25] and AOCS Ce8-89 [20].

#### **2.5. Oil-in-Water (O/W) emulsions preparation**

Refined sunflower oil was used to prepare oil-in-water (O/W) emulsions with a formula‐ tion of 30:70 wt/wt. 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, according to Cabezas et al. [26] with the addition of MSLs in a range of 0.1-2.0% (wt/wt).

#### **2.6. Optical characterization of emulsions**

The backscattering of light was measured using a QuickScan Vertical Scan Analyzer (Coult‐ er Corp., Miami, FL). The backscattering of monochromatic light (λ = 850 nm) from the emulsions was determined as a function of the height of the sample tube (ca. 65 mm) in or‐ der to quantify the rate of different destabilization processes during 60 min. This methodol‐ ogy allowed to discriminate between particle migration (sedimentation, creaming) and particle size variation (flocculation, coalescence) processes [27]. The basis of the multiple light scattering theory has been exhaustively studied by Mengual et al. [28].

#### **2.7. Statistical analysis**

Data were evaluated by analysis of variance (ANOVA) using the software Systat® 12.0 [29]. For this purpose, differences were considered significant at *p* <0.05.

## **3. Results**

**2.3. Phospholipid composition**

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**2.4. Antioxidant properties**

(wt/wt).

duction time, according to Gutiérrez [24].

ous heat treatments were used as control samples.

**2.5. Oil-in-Water (O/W) emulsions preparation**

**2.6. Optical characterization of emulsions**

Phospholipid composition of samples obtained after different modification processes was determined by 31P NMR analysis in a Bruker Avance 600 MHz automatic spectrometer, us‐ ing triphenyl phosphate as internal standard (Spectral Service GmbH, Köln, Germany) (Fig‐ ure 2) [21-23]. For this purpose, 100 mg of each modified lecithin were diluted in 1 mL of deuterated chloroform, 1 mL of methanol and 1 mL of 0.2 M Cs-EDTA (pH 8.0). The organic layer was separated after 15 min shaking, and analyzed by the described spectroscopic tech‐ nique. Phospholipid composition of the different modified sunflower lecithins (MSLs) was

The antioxidant properties of the MSLs were evaluated by the Rancimat (Mod 679, Met‐ rohm) method. 5 g of sunflower oil were added with different concentration of the analyzed samples (500-2000 ppm), heated at 98 °C, air flow 20 L/h. Stability was expressed as the in‐

Also, the highest antioxidant concentration was selected for each modified lecithin. The same procedure was carried out with previous thermal treatments at 120 and 160 °C for 1 h, adding the modified lecithins before heating. Refined sunflower oil with and without previ‐

Oil tocopherol content was determined by normal phase HPLC using a Hewlett Packard chromatography system (HPLC Hewlett Packard 1050 Series, Waldbronn, Germany) equip‐ ped with a fluorescence detector Agilent 1100 Series (Agilent Technology, Palo Alto, CA,

Refined sunflower oil was used to prepare oil-in-water (O/W) emulsions with a formula‐ tion of 30:70 wt/wt. 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, according to Cabezas et al. [26] with the addition of MSLs in a range of 0.1-2.0%

The backscattering of light was measured using a QuickScan Vertical Scan Analyzer (Coult‐ er Corp., Miami, FL). The backscattering of monochromatic light (λ = 850 nm) from the emulsions was determined as a function of the height of the sample tube (ca. 65 mm) in or‐ der to quantify the rate of different destabilization processes during 60 min. This methodol‐ ogy allowed to discriminate between particle migration (sedimentation, creaming) and particle size variation (flocculation, coalescence) processes [27]. The basis of the multiple

light scattering theory has been exhaustively studied by Mengual et al. [28].

USA) following the procedures described in IUPAC 2.432 [25] and AOCS Ce8-89 [20].

expressed in terms of molar concentration (mol / 100 mol lecithin) [17].

#### **3.1. Phospholipid composition**

The quantitative analysis of phospholipids was performed by 31PNMR which represents a modern and the most sophisticated methodology for evaluating the composition of lecithins, since it is possible to obtain a separate signal for each phospholipid class [9, 21]. The results pre‐ sented in Table 1, evidenced the high solubility of the phosphatidylcholine in the different ethanolic solvents assayed. 31PNMR determinations of the different enriched PC fractions (PCF 96 and PCF 100) exhibited an important concentration of PC (>71.0 %) as well as low values of PI (<8%) in comparison with the native and deoiled sunflower lecithin assayed.

#### **3.2. Rancimat analysis**

The oxidative stability of the refined sunflower oil (control) and the activity of different modified lecithins were evaluated determining the induction time (ti ) by a Rancimat analy‐ sis. This methodology can be used to evaluate the efficiency of various synthetic or natural antioxidants to stabilize fats and oils against an accelerated oxidation test [30]. Figure 3 shows ti for the modified sunflower lecithins used at different concentrations.


a Values represent means (n = 2). The coefficient of variation was lower than 6%

**Table 1.** Percentage phospholipid composition (mol PL / mol total PL) of deoiled sunflower lecithin and PC-enriched fractions analyzed by 31PNMR

The antioxidant addition increased the ti of the control as a function of increasing concentra‐ tion. MSLs did not show a marked difference of the respective ti values at concentration of 500 ppm. However, at high concentrations (1000 - 2000 ppm), both PC fractions had a high significant effect (*p* <0.01) on the oxidative stability of the control system, in relation to the values recorded by DSL addition. In this sense, 2000 ppm of DSL increased the ti value of the control oil of 49.1%, while similar concentrations of PCF 96 and PCF 100 enhanced this value 167.4% and 108.3%, respectively (Table 2).

troduction of changes in the original phospholipids concentration of this native lecithin by different modification processes (deoiling, ethanol fractionation) allow to obtain modified lecithins with better physicochemical and functional properties with respect to the starting

Antioxidant and Emulsifying Properties of Modified Sunflower Lecithin by Fractionation with Ethanol-Water Mixtures

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A concentration of 2000 ppm of the different modified lecithins showed the highest anti‐ oxidant activity in this assay. Therefore, refined sunflower oil added with this concentra‐ tion of MSLs was previously subjected to different heat treatments at 120 and 160 ° C for 1 h. The ti values of the oils added with PCF samples had a high significant difference (*p* <0.01) in relation to those added with DSL (Table 2). PC enriched fractions reduced the negative effect of the heat treatments over the oxidative stability of refined sunflower oil. These pre-treated samples showed higher ti values respect to the control system, even without thermal pre-treatment. In this sense, oils added with PCF 96 and PCF 100, sub‐ jected to a heat treatment at 160°C presented induction times of 58.6 and 44.9% higher

It is interesting to note that PCF 96 recorded the best antioxidant capacity producing an in‐ crease of 211.9% (120°C, 1h) and 113.9% (160°C, 1h) in relation to those recorded for the re‐ spective control oils. This fact indicates that the fractionation process carried out with ethanol 96 ° not only allows to obtain fractions enriched in PC with antioxidant activity, but also this activity is greater than those exhibited using DSL and the PCF obtained through

The ability of phospholipids to inhibit lipid oxidation in bulk oils has been known for sever‐ al decades, but the mechanism of stabilization still remains controversial [32]. However, many research works have proposed different antioxidant mechanisms for these com‐ pounds. Particularly, PC and PE have been shown to have metal-chelating and scavenging properties. Also, this type of phospholipids present a synergistic effect with the different to‐ copherols (α-tocopherol 512.84 µg/g; β-tocopherol 4.55 µg/g) regenerating the oxidized toco‐ pherol molecule by donation of a hydrogen atom of their amino function [18, 33]. This fact and the high PC and PE concentrations are in correlation with the better antioxidants char‐

Stability of the different O/W emulsions (30:70 wt/wt) was studied recording the backscat‐ tering (BS) profiles as a function of the cell length and time, by a vertical scan analyzer (QuickScan). These profiles were analysed in different zones of the emulsion: Zone I (10-20 mm) to visualize the destabilization process by migration of the oil droplets from the bottom towards the top of the tube (creaming), Zone II (40-45 mm) characterized by the accumula‐ tion of oil droplets after the creaming process (cream phase) and Zone III (50-60 mm), to an‐ alyze the destabilization of the cream phase [34]. For instance, Figure 4 shows a typical

profile obtained for an O/W emulsion with the addition of 0.5% of DSL.

material.

than the untreated oil, respectively.

fractionation with absolute ethanol (PCF 100).

acteristics observed for both PCF assayed.

**3.3. Optical characterization of O/W emulsions**

**Figure 3.** Induction times (ti) of refined sunflower oil (control) added with different concentrations of modified sun‐ flower lecithins (Rancimat Mod 679, Metrohm). Mean values (n = 3) ± sd


control, refined sunflower oil; WT, samples without thermal pre-treatment; T120, samples with thermal pre-treatment 120°C, 1h; T160, samples with thermal pre-treatment 160°C, 1h

**Table 2.** Percentage induction times increase of refined sunflower oil added with different modified sunflower lecithins (MSLs) with or without thermal pre-treatment

Induction times of oils added with the different MSLs without thermal pre-treatments, showed higher values than those obtained by Pan et al. [31], who reported under similar test conditions that 2000 ppm of native sunflower lecithin produced a ti increase of 12%. The in‐ troduction of changes in the original phospholipids concentration of this native lecithin by different modification processes (deoiling, ethanol fractionation) allow to obtain modified lecithins with better physicochemical and functional properties with respect to the starting material.

A concentration of 2000 ppm of the different modified lecithins showed the highest anti‐ oxidant activity in this assay. Therefore, refined sunflower oil added with this concentra‐ tion of MSLs was previously subjected to different heat treatments at 120 and 160 ° C for 1 h. The ti values of the oils added with PCF samples had a high significant difference (*p* <0.01) in relation to those added with DSL (Table 2). PC enriched fractions reduced the negative effect of the heat treatments over the oxidative stability of refined sunflower oil. These pre-treated samples showed higher ti values respect to the control system, even without thermal pre-treatment. In this sense, oils added with PCF 96 and PCF 100, sub‐ jected to a heat treatment at 160°C presented induction times of 58.6 and 44.9% higher than the untreated oil, respectively.

It is interesting to note that PCF 96 recorded the best antioxidant capacity producing an in‐ crease of 211.9% (120°C, 1h) and 113.9% (160°C, 1h) in relation to those recorded for the re‐ spective control oils. This fact indicates that the fractionation process carried out with ethanol 96 ° not only allows to obtain fractions enriched in PC with antioxidant activity, but also this activity is greater than those exhibited using DSL and the PCF obtained through fractionation with absolute ethanol (PCF 100).

The ability of phospholipids to inhibit lipid oxidation in bulk oils has been known for sever‐ al decades, but the mechanism of stabilization still remains controversial [32]. However, many research works have proposed different antioxidant mechanisms for these com‐ pounds. Particularly, PC and PE have been shown to have metal-chelating and scavenging properties. Also, this type of phospholipids present a synergistic effect with the different to‐ copherols (α-tocopherol 512.84 µg/g; β-tocopherol 4.55 µg/g) regenerating the oxidized toco‐ pherol molecule by donation of a hydrogen atom of their amino function [18, 33]. This fact and the high PC and PE concentrations are in correlation with the better antioxidants char‐ acteristics observed for both PCF assayed.

#### **3.3. Optical characterization of O/W emulsions**

The antioxidant addition increased the ti of the control as a function of increasing concentra‐ tion. MSLs did not show a marked difference of the respective ti values at concentration of 500 ppm. However, at high concentrations (1000 - 2000 ppm), both PC fractions had a high significant effect (*p* <0.01) on the oxidative stability of the control system, in relation to the

the control oil of 49.1%, while similar concentrations of PCF 96 and PCF 100 enhanced this

**Figure 3.** Induction times (ti) of refined sunflower oil (control) added with different concentrations of modified sun‐

Control 0.0 0.0 0.0 -26.6 -25.8 DSL 49.1 56.9 19.0 15.1 -11.7 PCF 96 167.4 211.9 113.9 128.8 58.6 PCF 100 157.7 177.5 95.4 103.6 44.9

control, refined sunflower oil; WT, samples without thermal pre-treatment; T120, samples with thermal pre-treatment

Induction times of oils added with the different MSLs without thermal pre-treatments, showed higher values than those obtained by Pan et al. [31], who reported under similar test conditions that 2000 ppm of native sunflower lecithin produced a ti increase of 12%. The in‐

**Table 2.** Percentage induction times increase of refined sunflower oil added with different modified sunflower

**Δ ti T160 / ti control T160 (%)**

**Δ ti T120 / ti control WT (%)**

**Δ ti T160 / ti control WT (%)**

value of

values recorded by DSL addition. In this sense, 2000 ppm of DSL increased the ti

value 167.4% and 108.3%, respectively (Table 2).

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flower lecithins (Rancimat Mod 679, Metrohm). Mean values (n = 3) ± sd

120°C, 1h; T160, samples with thermal pre-treatment 160°C, 1h

lecithins (MSLs) with or without thermal pre-treatment

**Δ ti T120 / ti control T120 (%)**

**Δ ti WT / ti control WT (%)**

> Stability of the different O/W emulsions (30:70 wt/wt) was studied recording the backscat‐ tering (BS) profiles as a function of the cell length and time, by a vertical scan analyzer (QuickScan). These profiles were analysed in different zones of the emulsion: Zone I (10-20 mm) to visualize the destabilization process by migration of the oil droplets from the bottom towards the top of the tube (creaming), Zone II (40-45 mm) characterized by the accumula‐ tion of oil droplets after the creaming process (cream phase) and Zone III (50-60 mm), to an‐ alyze the destabilization of the cream phase [34]. For instance, Figure 4 shows a typical profile obtained for an O/W emulsion with the addition of 0.5% of DSL.

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 and the appear‐ ance of an isosbestic point [34]. This point separates the zones with lower (left) and higher (right) values than the initial %BS (Figure 3). The QuickScan profiles corresponding to the zone I (10-20 mm) showed an increase of the emulsion stability against the creaming proc‐ ess, as a function of increasing concentration of different modified lecithins (Figure 5). In particular, the PC enriched fractions (PCF 96 and PCF 100) produced a high stability in O/W emulsions than DSL, over the range of concentration studied. In this sense, it should be not‐ ed that QuickScan profiles of the different PCF did not show significant variations of %BS values for 2.0% during 40 minutes. These results are in concordance with those previously reported by Wu and Wang [12], related to the emulsifying activity of PC enriched fractions

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Moreover, O/W emulsions with 0.1-0.5% of DSL showed a sharp decrease of %BS in the Zone I. This behaviour indicates a rapid destabilization of these emulsions by creaming.

**Figure 6.** Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sun‐

flower lecithins, Zone II (40-45 mm). Mean values (n = 3) ± sd

from soy lecithin.

**Figure 4.** Backscattering (%BS) profile of a O/W emulsion (30:70 wt/wt) with the addition of 0.5% of DSL

**Figure 5.** Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sun‐ flower lecithins, 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 and the appear‐ ance of an isosbestic point [34]. This point separates the zones with lower (left) and higher (right) values than the initial %BS (Figure 3). The QuickScan profiles corresponding to the zone I (10-20 mm) showed an increase of the emulsion stability against the creaming proc‐ ess, as a function of increasing concentration of different modified lecithins (Figure 5). In particular, the PC enriched fractions (PCF 96 and PCF 100) produced a high stability in O/W emulsions than DSL, over the range of concentration studied. In this sense, it should be not‐ ed that QuickScan profiles of the different PCF did not show significant variations of %BS values for 2.0% during 40 minutes. These results are in concordance with those previously reported by Wu and Wang [12], related to the emulsifying activity of PC enriched fractions from soy lecithin.

Moreover, O/W emulsions with 0.1-0.5% of DSL showed a sharp decrease of %BS in the Zone I. This behaviour indicates a rapid destabilization of these emulsions by creaming.

**Figure 4.** Backscattering (%BS) profile of a O/W emulsion (30:70 wt/wt) with the addition of 0.5% of DSL

**Figure 5.** Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sun‐

flower lecithins, Zone I (10-20 mm). Mean values (n = 3) ± sd

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**Figure 6.** Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sun‐ flower lecithins, Zone II (40-45 mm). Mean values (n = 3) ± sd

The tube zone between 40-45 mm (Zone II) is characterized by the accumulation of oil drop‐ lets after the creaming process (cream phase). Figure 6 shows the %BS values vs. time in Zone II. Emulsions formulated with PC enriched fractions 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 formulated with a high concentration of phosphati‐ dylcholine would be associated with the formation of dense cream phases with a lower pro‐ portion of continuous phase inside [26].

However, emulsion with 0.1 of DSL did not allow the formation of the cream phase. These results are related to the rapid decrease of %BS, the formation of an oil layer in the upper part of the tube and the absence of an isosbestic point (data not shown) ; suggesting the oc‐

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The hydrophilic-lipophilic balance value (HLB) is often used in order to explain the per‐ formance of emulsifiers [15]. The high concentration of phosphatidylcholine (hydrophilic phospholipid) increases this empirical value in the PC enriched fractions. In this sense, the best properties of these modified lecithins as emulsifying agents in O/W emulsions was ac‐ cording with Carlsson [35], who has determined that lecithin with high HLB values present

Also, the phase structure at the interface of the different phospholipids influences the emul‐ sion formation and stability [16]. PC forms a lamellar phase at the interface between oil and water with well ordered mono- and bi- layers. This structure has a great importance for the

The study of the induction times showed a highly significant difference (*p* <0.01) between the antioxidant activity exhibited by the different PCF in relation to the addition of DSL. Al‐ so, these fractions allowed to obtain more stable O/W emulsions (30:70 wt/wt) in compari‐ son with those added with DSL at different concentrations (0.1-2.0%) in terms of kinetic destabilization as a function of changes in the backscattering values vs. time. These results showed that PC enriched fractions (PCF 96 and PCF 100) constitute a potential alternative as

This work was supported by grants from Agencia Nacional de Promoción Científica y Tec‐ nológica (ANPCyT), Argentina (PICT 2007-1085), Consejo Nacional de Investigaciones Cien‐ tíficas y Técnicas (CONICET), Argentina, PIP 1735 (CONICET); and Universidad Nacional

D. M. Cabezas and M. C. Tomás are members of the Career of Scientific and Technological Researcher of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ar‐ gentina. E. N. Guiotto is a recipient of a doctoral fellowship from Consejo Nacional de Inves‐ tigaciones Científicas y Técnicas (CONICET). B. W. K. Diehl is Director of Spectral Service

Sunflower lecithin was provided by Néstor Buseghin (Vicentin S.A.I.C., Argentina). Thors‐ ten Buchen and Rute Azevedo (Spectral Service, Germany) are acknowledged for technical

currence of a cream phase destabilization by coalescence [27].

best O/W emulsifying properties.

stabilisation of O/W emulsions.

emulsifier agent for the food industry.

de La Plata (UNLP), Argentina, 11/X502 (UNLP).

**Acknowledgments**

GmbH, Cologne, Germany.

assistance.

**4. Conclusions**

The stability of the different cream phases were analyzed by the evolution of the % BS val‐ ues vs. time in Zone III (50-60 mm) (Figure 7). %BS values remained constant at concentra‐ tions of emulsifier above 0.1% of the different modified lecithins. This behaviour confirms the stability provided by these MSLs against the coalescence process, in these conditions.

**Figure 7.** Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sun‐ flower lecithins, Zone III (50-60 mm). Mean values (n = 3) ± sd

However, emulsion with 0.1 of DSL did not allow the formation of the cream phase. These results are related to the rapid decrease of %BS, the formation of an oil layer in the upper part of the tube and the absence of an isosbestic point (data not shown) ; suggesting the oc‐ currence of a cream phase destabilization by coalescence [27].

The hydrophilic-lipophilic balance value (HLB) is often used in order to explain the per‐ formance of emulsifiers [15]. The high concentration of phosphatidylcholine (hydrophilic phospholipid) increases this empirical value in the PC enriched fractions. In this sense, the best properties of these modified lecithins as emulsifying agents in O/W emulsions was ac‐ cording with Carlsson [35], who has determined that lecithin with high HLB values present best O/W emulsifying properties.

Also, the phase structure at the interface of the different phospholipids influences the emul‐ sion formation and stability [16]. PC forms a lamellar phase at the interface between oil and water with well ordered mono- and bi- layers. This structure has a great importance for the stabilisation of O/W emulsions.

## **4. Conclusions**

The tube zone between 40-45 mm (Zone II) is characterized by the accumulation of oil drop‐ lets after the creaming process (cream phase). Figure 6 shows the %BS values vs. time in Zone II. Emulsions formulated with PC enriched fractions 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 formulated with a high concentration of phosphati‐ dylcholine would be associated with the formation of dense cream phases with a lower pro‐

The stability of the different cream phases were analyzed by the evolution of the % BS val‐ ues vs. time in Zone III (50-60 mm) (Figure 7). %BS values remained constant at concentra‐ tions of emulsifier above 0.1% of the different modified lecithins. This behaviour confirms the stability provided by these MSLs against the coalescence process, in these conditions.

**Figure 7.** Backscattering (%BS) values of O/W emulsions (30:70 wt/wt) with the addition of different modified sun‐

flower lecithins, Zone III (50-60 mm). Mean values (n = 3) ± sd

portion of continuous phase inside [26].

598 Food Industry

The study of the induction times showed a highly significant difference (*p* <0.01) between the antioxidant activity exhibited by the different PCF in relation to the addition of DSL. Al‐ so, these fractions allowed to obtain more stable O/W emulsions (30:70 wt/wt) in compari‐ son with those added with DSL at different concentrations (0.1-2.0%) in terms of kinetic destabilization as a function of changes in the backscattering values vs. time. These results showed that PC enriched fractions (PCF 96 and PCF 100) constitute a potential alternative as emulsifier agent for the food industry.

## **Acknowledgments**

This work was supported by grants from Agencia Nacional de Promoción Científica y Tec‐ nológica (ANPCyT), Argentina (PICT 2007-1085), Consejo Nacional de Investigaciones Cien‐ tíficas y Técnicas (CONICET), Argentina, PIP 1735 (CONICET); and Universidad Nacional de La Plata (UNLP), Argentina, 11/X502 (UNLP).

D. M. Cabezas and M. C. Tomás are members of the Career of Scientific and Technological Researcher of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ar‐ gentina. E. N. Guiotto is a recipient of a doctoral fellowship from Consejo Nacional de Inves‐ tigaciones Científicas y Técnicas (CONICET). B. W. K. Diehl is Director of Spectral Service GmbH, Cologne, Germany.

Sunflower lecithin was provided by Néstor Buseghin (Vicentin S.A.I.C., Argentina). Thors‐ ten Buchen and Rute Azevedo (Spectral Service, Germany) are acknowledged for technical assistance.

## **Author details**

Dario M. Cabezas1 , Estefanía N. Guiotto1 , Bernd W. K. Diehl2 and Mabel C. Tomás1\*

\*Address all correspondence to: mabtom@hotmail.com

1 Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA) – CCT La Plata – CONICET - Facultad de Ciencias Exactas, Universidad Nacional de La Plata (FCE - UNLP), La Plata, Argentina

[10] van Nieuwenhuyzen W. Fractionation of Lecithins. The European Food and Drink

Antioxidant and Emulsifying Properties of Modified Sunflower Lecithin by Fractionation with Ethanol-Water Mixtures

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[14] Hernández E, Quezada N. Uses of phospholipids as functional ingredients. In: Gun‐ stone FD (ed.) Phospholipid Technology and Applications. Bridgwater: The Oily

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[16] van Nieuwenhuyzen W, Tomás MC. Update on vegetable lecithin and phospholipid technologies. European Journal of Lipid Science and Technology 2008; 110, 472-486.

[17] Cabezas DM, Diehl BWK, Tomás MC. Effect of processing parameters on sunflower PC enriched fractions extracted with aqueous-ethanol. European Journal of Lipid Sci‐

[18] Saito H, Ishihara K. Antioxidant activity and active sites of phospholipids as antioxi‐

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Dario M. Cabezas1

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**Chapter 26**

**Cabannina Cattle Breeding: An Agro-Ecological**

**Challenge for Sustainable Rural Development in**

Intensive farming is an agricultural production system characterized by widely adopting ex‐ ternal inputs - such as capital, mechanization, infrastructures, pesticides and chemical fertil‐ izers intensively used - which affects the natural environment and rural societies. Since it allows to produce more food on a given land extension, such agricultural choice has been the predominant response to population growth so far. While permitting to raise many ani‐ mals in limited areas, intensive animal farming practices require a large amount of food, wa‐ ter, medical treatments, capital intensive technology, energy, and fuel. Is being the selection of animals with rapid food conversion into milk and meat the aim of every industrial farm, a decline in, for example, the animal reproductive performances and in the product quality follows. Thus, nowadays problems in the dairy cattle scenario are easily highlighted. Just to name Friesian breed, its reproductive performances decreased worldwide with negative consequences on both cow robustness and longevity due to increased stress, udder health disturbances and locomotion disorders, which meant damages to the physiological parame‐

Despite all the above mentioned problems associated with conventional farming, many pos‐ itive developments are creeping in. Several alternative initiatives are now flourishing all around the Italian peninsula to promote ecological agriculture; preservation of small farm‐ ers' livelihoods; production of healthy, safe and tradition-linked foods; localization of distri‐ bution, trade and marketing. These typologies of traditional agriculture offer promising models for marginal areas as they promote biodiversity, thrive without agrochemicals, and

and reproduction in any medium, provided the original work is properly cited.

© 2013 Communod et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Northern Italy**

http://dx.doi.org/10.5772/53155

ters typical of healthy cows.

**1. Introduction**

Ricardo Communod, Carla Colombani, Eleonora Munari and Daniele Vigo

Additional information is available at the end of the chapter
