Soybean Proteins by Response Surface

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Effect of extrusion on the emulsifying properties of soybean proteinsand pectin mixtures modelled by response surface methodology

Arthur Soares Bueno a, Cristina Meyer Pereira a, Bruna Menegassi b, José Alfredo Gomes Arêas b,Inar Alves Castro a,*

a Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Lineu Prestes 580 B14, 05508-900 São Paulo, Brazilb Department of Nutrition, Faculty of Public Health, University of São Paulo, Av. Dr. Arnaldo, 715, 05508-900 São Paulo, Brazil

a r t i c l e i n f o

Article history:

Received 3 March 2008

Received in revised form 17 July 2008

Accepted 24 July 2008

Available online 3 August 2008

Keywords:

Pectin

Soybeans

Hydrocolloid

Protein

Emulsion

Extrusion

RSM

a b s t r a c t

The objectiveof this study was to applyresponse surface methodology to estimatethe emulsifying capac-

ity and stability of mixtures containing isolated and textured soybean proteins combined with pectin and

to evaluate if the extrusion process affects these interfacial properties. A simplex-centroid design was

applied to the model emulsifying activity index (EAI), average droplet size (D[4,3]) and creaming inhibition

(CI%) of the mixtures. All models were significant and able to explain more than 86% of the variation. The

high predictive capacity of the models was also confirmed. The mean values for EAI, D[4,3] and CI%

observed in all assays were 0.173 ± 0.015 nm, 19.2 ± 1.0lm and 53.3 ± 2.6%, respectively. No synergism

was observedbetween thethree compounds. This result canbe attributed to thelow soybean protein sol-

ubility at pH 6.2 (<35%). Pectin was the most important variable for improving all responses. The emul-

sifying capacity of the mixture increased 41% after extrusion. Our results showed that pectin could

substitute or improve the emulsifying properties of the soybean proteins and that the extrusion brings

additional advantage to interfacial properties of this combination.

2008 Elsevier Ltd. All rights reserved.

1. Introduction

One of the biggest challenges for food scientists is to develop

new emulsifiers, preferably using natural compounds, that are able

to produce a stable film around the small oil droplets during the

emulsion process. The use of chemical stabilizers in the food indus-

try is not desirable, and health authorities in many countries are

constantly enforcing restrictions and limitations on the use of syn-

thetic emulsifiers (Benichou et al., 2002; Dalev and Simeonova,

2006; Mun et al., 2008). Natural compounds, such as some phos-

pholipids, proteins and high molecular weight hydrocolloids, have

been applied for this purpose (Edris, 1998; Scuriatti et al., 2003).

Proteins constitute an important group of emulsifier agents used

in foods because of their surface-active properties, which are a

consequence of their conformation. Surface hydrophobicity and

solubility are the major factors determining emulsifying activity,

while the molecular flexibility of the proteins is important for their

emulsion stability (Edris, 1998; Benichou et al., 2002; Damodaran,

2005). Soybean proteins aid in the formation of emulsions, mainly

by decreasing the interfacial tension between water and oil and

they also help to stabilize the emulsion by forming a physical bar-

rier at the interface (Molina et al., 2001). Soybean proteins, isolate

or textured, have been widely applied as emulsifiers in food

products.

Polysaccharides are often used to improve the stability of food

emulsions (Nakamura et al., 2004; Liu et al., 2008). Water-soluble

polysaccharides, often termed hydrocolloids or gums, have little

surface activity. Hydrocolloids stabilize emulsions by increasing

the viscosity of the continuous phase. Consequently, collisions be-

tween the droplets of the dispersed phase are less frequent, delay-

ing the phase separation (Cameron et al., 1991; Huang et al., 2001;

Benichou et al., 2002; Makri et al., 2005). Some natural food hydro-

colloids, including pectin, gum arabic, microcrystalline-cellulose,

galactomannans and soluble soybean polysaccharides, exhibit

emulsifying properties due to the mechanic stabilization effects.

This proteinaceous moiety, which is covalently bound to a high

weight fraction of the carbohydrate backbone, adsorbs onto the

oil–water interface as an anchor (Nakamura et al., 2004; Akhtar

et al., 2002; Leroux et al.,2003; Funami et al., 2007). Pectins are

semi-flexible polymers of D-galacturonic acid linked together

through a-1, 4-glycosidic linkages interrupted by L -rhamnose res-

idues (Akhtar et al., 2002). Some of the carboxylic groups in the

pectin chains are methyl esterified. Depending on the degree of

esterification (DE), pectins are divided into two major groups: high

methoxyl pectin (HMP) with a DE higher than 50% and low meth-

oxyl pectin (LMP) with a DE lower than 50% ( Leroux et al., 2003).

The proportion of ester groups and the molecular mass influence

0260-8774/$ - see front matter 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.jfoodeng.2008.07.028

* Corresponding author. Tel.: +55 11 30911481; fax: +55 11 38154410.

E-mail address: [email protected] (I.A. Castro).

Journal of Food Engineering 90 (2009) 504–510

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the emulsification properties of pectins (Drusch, 2007). According

to Leroux et al. (2003), citrus pectin with a low molecular weight

and a high degree of methoxylation (>70%) showed a high emulsi-

fying property and was able to stabilize oil in a water emulsion.

Protein–polysaccharide interactions have been extensively studied

because they appear in most food systems (Benichou et al., 2002).

Many studies have reported that synergistic interaction occurs in

food emulsion formation as well as emulsion stability when pro-

teins are mixed with polysaccharides (Uruakpa and Arntfield,

2005; Makri et al., 2005). Proteins are natural polymeric surfac-

tants and typically form an adsorbed primary layer at the oil–water

interface, whereas, hydrophilic polysaccharides may form a thick

secondary layer, which enhances the steric stabilizing properties

on the outside of the protein-coated droplets (Leal-Calderon

et al., 2007; Nakamura et al., 2006; Benichou et al., 2002 ).

Response surface methodology (RSM) is a collection of mathe-

matical and statistical techniques useful for developing, improving,

and optimizing processes (Myers and Montgomery, 2002). RSM of-

fers a large amount of information from a small number of exper-

iments, allows for the observation of the interaction effect of the

independent parameters on the response, and is a useful tool for

the optimization of chemical and biochemical processes (Bas and

Boyaci, 2007). A mixture experiment is a special type of response

surface experiment in which the factors are the components of a

mixture, and the response is a function of the proportions of each

component. The canonical form of the most complex mixture mod-

el (special cubic) for a mixture containing three components can be

given as: E(y) =Rbi xi + bij xi x j + bijk xix jxk, where bi are the model

coefficients, xi the variables and E ( y) the estimative of the response

(Myers and Montgomery, 2002).

Moderate heat treatment such as extrusion, which employs

high temperatures over short times, promotes partial protein

denaturation and starch gelatinization. The thermal process alters

the distribution pattern of hydrophobic and hydrophilic patches on

the protein surfaces, consequently affecting their interfacial prop-

erties (Damodaran, 1996). Limited studies have been performed

on the comparison of the influence of ionic hydrocolloids in pro-tein–polysaccharide systems (Ercelebi and Ibanoglu, 2007). Based

on the fact that proteins and polysaccharides could exert a comple-

mentary function as emulsifiers in food formulations, depending

on the type and proportion of each component, our objective was

to apply the RSM technique to estimate the emulsifying properties

of the mixture containing soybean proteins and high methoxyl

pectin and to evaluate the extrusion process influence on these

properties.

2. Materials and methods

2.1. Materials

Soybean protein isolated (Maisol) and textured soybean protein

(NL1000) were provided by Exin Ind. Com. Ltd. (Massaranduba,

Brasil). Highly methyl esterified pectin extracted from citrus peel

(GrindstedPectin USP) was provided by Danisco Cultor Brasil

Ltd. All used reagents were of analytical grade.

2.2. Chemical composition of the ingredients

The chemical composition of ingredients, such as the moisture,

fat, protein and ash contents, were determined by the method de-

scribed in AOAC (1990). The carbohydrate content was obtained by

difference. The dietary fiber of the ingredients was analyzed by an

enzymatic and gravimetric method modified by Prosky et al.

(1985). Quadruplicate samples (1 g) were digested with thermo-stable alpha-amylase (pH 6.0), protease (pH 7.5) and amylogluco-

sidase (pH 4.3) to remove protein and starch. The hydrolysate

was vacuum-filtered using crucibles pre-washed with an extra

solution and glass wool to separate the soluble fraction from the

insoluble one. Four volumes of 98% ethanol were added to precip-

itate the soluble dietary fiber. The residue was filtered; washed

first with 78% ethanol and then with 95% ethanol and acetone;

and finally dried and weighed. A duplicate was analyzed for pro-

tein, and another was incinerated at 525 C for ash determination.

2.3. Protein solubility determination

The protein solubility of the samples was determined using the

method of Dyer-Hurdon and Nnanna (1993), according to which

10 mL of the solutions at pH 6.2 (1%, w/v) were centrifuged

(10.000 g ) for 30min at 20 C; being the protein content of the

supernatant determined by the Lowry method, using BSA (Sigma

A7906) as the standard (Lowry et al.,1951).

2.4. Experimental design and statistical analysis

A simplex-centroid design was applied to model the emulsifying

properties of the mixtures containing three ingredients: citrus pec-

tin (PEC, x1), textured soybean protein (TSP, x2) and soybean pro-

tein isolate (SPI, x3). Three additional points were included in the

design for the further predictive capacity analysis of the models.

Residual analysis, the coefficient of determination (adjusted R2),

the significance of the models and the lack of fit were used to check

the quality of the model. The results from all parameters were

evaluated to check the homogeneity of the variances (Hartley F max)

and submitted to one-way ANOVA, because these assumptions had

to be tested before the RSM analysis. The results were expressed as

mean ± SD. A chi-square statistical test (v2) was applied to com-

pare the observed values with the values predicted by the models.

The T -test for dependent samples was used to compare the data of

the protein–pectin mixture before and after extrusion. An a value

of 0.05 was fixed, and calculations were performed using the Stat-

istica version 7.1 software (Statsoft Inc., Tulsa, OK, USA). The re-sults presented are the averages of two complete and

independent experiments.

2.5. Extrusion process

Extrusion experiments were carried out in a laboratory single

screw extruder (RXPQ Labor 24, Inbramak Ind. Maq. Ltd., Ribeirão

Preto, Brazil). The barrel had three zones with independent electric

resistance heaters and a 3.55:1 compression ratio screw. The tem-

perature in the barrel from the inlet to the die was set at 50 C,

115 C and 135 C, with a moisture content of 15% and a screw

speed rotation of 263 rpm. The die diameter adopted was

4.68 mm. The feed mass flow rate was kept constant at 100 g/

min (dry matter). The temperatures of all the sections were set,and, once these were reached, corn grits were extruded at a screw

speed of 263 rpm to stabilize the flow at 200 g/min before pro-

cessing the soy proteins and pectin mixture. Finally, the mixture

was fed to the extruder, and, after 5 min and stable ampere input

readings, the samples were collected. All processing conditions

were controlled using software for Windows developed in the lab-

oratory of the Faculty of Public Health of University of São Paulo

(São Paulo, Brasil). After extrusion, the samples were cooled down

to room temperature, milled and stored in polyethylene bags for

further analysis.

2.6. Emulsion activity index (EAI)

Ingredients (1 g) were dissolved in 50 mL of distilled water, andthe pH of the solutions was adjusted to 6.2 (Makri et al., 2005). The

A.S. Bueno et al. / Journal of Food Engineering 90 (2009) 504–510 505



solutions were then stirred for 60 min at room temperature on a

magnetic stirring plate, and the volume was adjusted to 100 mL.

The oil/water emulsions were prepared by adding 5 mL of soybean

oil to a 15 mL sample solution (1%, w/v). The crude emulsion was

homogenized for 90 s using a mixer (Tecnal TE-102, Piracicaba)

at 16.000 rpm. The emulsifying activity index (EAI) was deter-

mined by the spectroturbidimetric procedure proposed by Pearce

and Kinsela (1978). Briefly, a 30l

L aliquot of the emulsion was re-

moved and diluted with 20 mL of 0.1% sodium dodecyl sulfate. The

absorbance was measured at 500 nm in a SHIMADZU UV1240 (Shi-

madzu Corporation, Tokyo, Japan) spectrophotometer using a 0.1%

SDS solution as a blank. In the spectrophotometer, where none of

the light scattered by the turbid sample reaches the photodetector

for a sample which does not absorb light, the turbidimetry is given

by T = (2.303 A)/L, where A is the observed absorbance and L is the

path length of the cuvette. For a dilute dispersion interfacial,

A = 2T. By this method EAI is calculated as EAI = 2T/Ø C, where Ø

is the volume fraction of the dispersed phase and C is the weight

of protein per unit volume of the aqueous phase before the emul-

sion is formed. In our study, since there were solutions with very

low protein content and the Ø value was the same for all samples,

EAI was expressed directly as the absorbance value taken at

500 nm, as reported by Chung and Ferrier (1991), since EAI is di-

rectly related to the interfacial area of the emulsion.

2.7. Droplet size distribution

The droplet size distribution of the emulsion was determined by

integrated light scattering using a Malvern Mastersizer S-MAM

5005 (Malvern Instruments Ltda, Malvern, UK) equipped with a li-

quid dispersion tank. The light (laser, He/Ne, k = 633 nm) is spread

by the emulsion sample, the droplet particle size being inverse to

the angle deviation. Measurements were performed at room tem-

perature (20 ± 2 C). The obscuration in all the measurements

was about 15%, and the relative refractive index of the lipid phase

to water and the absorption were set at 1.15 and 0.1, respectively.

The average droplet size of the emulsions (25 vol%, pH 6.2) wascharacterized by D[4,3], which represents the mean diameter

weighed in volume: D[4,3] =Ri nidi4/Rinidi

3 (lm), where ni is the

number of the droplets of diameter di. Ingredients which produce

a lower D[4,3] index seem to be more effective emulsifiers (Leroux

et al., 2003).

2.8. Emulsion stability (ES)

Fresh emulsions were prepared as described above and poured

into a graduated cylinder. The destabilization was followed by

plotting the volume of the remaining emulsion phase as a function

of time at room temperature according to Chung and Ferrier

(1991). The heights of the lower aqueous layer, middle (emulsion)

layer and the upper oil layer were recorded to the nearest 0.5 mm.The creaming inhibition (CI) was calculated as the percentage of

the emulsion layer remaining after 24 h compared to the freshly

made emulsion, CI = [1(H t/H o)] 100. The samples were ana-

lyzed in triplicates.

2.9. Light microscopy

The microstructure of the emulsions was examined using light

microscopy immediately after preparing the emulsion under bright

field illumination with a 5–50 objective lens equipped with a Lei-

ca Microscope (DMLM, Germany) able to increase the image size

500, with an adapted digital camera. A drop of emulsion was

placed on a microscope slide and then covered with a cover slip.

The procedure was duplicated by taking a second sample from

the same emulsion after 24 h of storage at room temperature

(20±2 C). By the photomicrographs it is possible to observe the

state of droplet flocculation and also to confirm the droplet size

distribution.

3. Results

Table 1 shows the chemical composition of the ingredients. Iso-

lated soybean protein was characterized by its high protein con-

tent (82.5%), while textured soybean protein presented higher

insoluble fiber content (21.1%). The pectin sample applied in this

study showed a significant protein proportion (5.7%) and a high

soluble fiber content (72.0%). The three-factor simplex-centroid

experimental design and the emulsifying properties evaluated in

this study are shown in Table 2. The different proportions between

proteins and polysaccharides caused significant alteration in all

measurements ( p < 0.01). The coefficients of the polynomial mod-

els and their respective quality analysis are presented in Table 3.

All models were significant ( p < 0.01) and able to explain 86–98%

of the variation (adjusted R2). No significant lack of fit was ob-

served ( p > 0.05), showing the excellent correlation between the

Table 1

Chemical composition of the ingredients

Nutrients (g/100g)a

Moisture Proteinb Lipids Ashes Carbohydratec Insoluble fiber Soluble fiber

Isolated soybean protein 8.3 ± 0.0 82.5 ± 3.6 0.3 ± 0.2 4.1 ± 0.8 4.0 ± 5.3 5.0 ± 0.0 2.4 ± 0.6

Textured soybean protein 3.3 ± 0.0 55.6 ± 3.1 1.1 ± 0.1 6.3 ± 0.0 33.0 ± 4.2 21.1 ± 0.6 2.1 ± 0.3

High methoxyl pectin 13.9 ± 0.0 5.7 ± 0.1 0.8 ± 0.1 2.9 ± 0.0 76.7 ± 0.1 0.1 ± 0.0 72.0 ± 2.7

a Values are mean± SD (n = 3).b

Calculated using N 6.25 for all samples.c Values obtained by difference.

Table 2

Responses to the samples prepared according to the three factor simplex-centroid

experimental design

Surfactant

mixture

Ingredient’s proportion

in the mixturea

Dependent variables or responses ( y)b

PEC

( x1)

TSP

( x2)

SPI

( x3)

EAI (nm) D[4,3]

(lm)

CI (%)

PEC 1.00 0.00 0.00 0.289 ± 0.014 9.3 ± 0.2 86.7 ± 5.8

TSP 0.00 1.00 0.00 0.150 ± 0.016 17.9 ± 1.6 69.5 ± 1.9

SPI 0.00 0.00 1.00 0.139 ± 0.023 20.3 ± 1.8 59.3 ± 2.1

PEC + TSP 0.50 0.50 0.00 0.192 ± 0.024 15.7 ± 0.5 65.3 ± 2.8

PEC + SPI 0.50 0.00 0.50 0.166 ± 0.012 23.1 ± 0.5 32.8 ± 3.6

TSP + SPI 0.00 0.50 0.50 0.128 ± 0.015 24.8 ± 1.6 29.8 ± 0.6

PEC + TSP + SPI 0.33 0.33 0.33 0.147 ± 0.003 23.4 ± 0.6 30.0 ± 1.5

pc – – – <0.01 <0.01 <0.01

a High methoxyl pectin (PEC), textured soybean protein (TSP) and isolated soy-

bean protein (ISP).b Values expressed as mean ± SD.c Probability value obtained by one-way ANOVA.

506 A.S. Bueno et al. / Journal of Food Engineering 90 (2009) 504–510



models and the experimental data. The high predictive capacity of

the models was confirmed in Table 4, where three additional

points were experimentally evaluated, and their results did not dif-

fer from the values estimated by the respective models ( p > 0.05). A

mixture containing isolated soybean protein, textured soybean

protein and pectin was prepared by mixing the three ingredients

(natural) in a specific proportion suggested by the optimization

of the model (data not shown). The mixture was then submittedto extrusion (extrused), and the chemical composition and emulsi-

fying properties of the two samples are shown in Table 5. Extrusion

improved the emulsifying capacity ( p < 0.01) of the mixture with-

out changing the stability ( p = 0.41). No differences were observed

in the soluble/insoluble fiber proportion between the two samples.

Contour plots generated from the fitted models for each re-

sponse are shown in Fig. 1. Pectin was the most important variable

to improve EAI and D[4,3] responses. At higher proportions of pectin

in the mixture, higher EAI and lower mean diameter of the droplets

(D[4,3]) were observed. The stability measured by the creaming

inhibition (CI) was also favored by the higher pectin proportion.

The light microscopy applied to the emulsions (Fig. 2) showed a

better homogeneity of the solution containing pectin than the

solution containing soybean proteins. This result could be partiallydue to the poor solubility of the soybean proteins at pH 6.2 (Fig. 3).

4. Discussion

The effect of each component of the mixture can be observed by

the magnitude, significance (Standard Error) and signal (+ or )

associated with the respective coefficient in the fitted model for

each response. The value and signal of the linear coefficients ob-

tained for each response (Table 3) showed that all components

contributed to increasing the emulsifying capacity (positive bi for

EAI), as well as the stability (positive bi for CI%), but also increased

the mean diameter of the oil droplet (positive bi for D[4,3]). The mix-

tures containing the highest proportion of pectin showed a better

emulsifying capacity, a better stability and the lowest mean diam-

eter of the oil droplet (D[4,3]), as corroborated in Fig. 2. This result

agrees with those reported by Akhtar et al. (2002), where the

authors concluded that pectin gave the best results in terms of par-

ticle diameter and stability in regards to creaming, these properties

being the ones affected by the molecular weight and the amount of

protein present.

The pectin’s emulsifying capacity and stability have been re-

ported by other studies. The proteinaceous component of the pec-

tin acts as an anchor at the oil–water interface, while the attached

polysaccharide chains provide the thick protective layer that con-

fers steric stabilization during extended storage (Akhtar et al.,

2002; Leroux et al., 2003; Williams et al., 2005). Leroux et al.

(2003) postulated that pectin is able to reduce the interfacial ten-

sion between oil and water and can be useful in the preparation of

emulsions. The authors observed that pectin at a 2% concentration

had an effect similar to that of gum arabic at 15% on the interfacial

tension reduction, giving better results in terms of the distribution

profile and mean diameter of the oil droplet. They attributed this

effect to the protein residues, the acetyl groups present withinthe pectin and the nature of the oil used in the emulsion. The drop-

let size can be smaller if the polysaccharide is present during

homogenization and stable emulsions can be obtained as the con-

centration of gums increases, since both high viscosity and small

oil droplet size contribute to emulsion stabilization (Huang et al.,

2001; Benichou et al., 2002; Funami et al., 2007; Ercelebi and

Ibanoglu, 2007). Contour curves obtained to CI responses (Fig. 1)

reinforced the significant and well-known influence of pectin on

emulsion stability (Leal-Calderon et al., 2007; Neirynck et al.,

2007).

Binary coefficients of the polynomial models (Table 3) showed

that all interactions were antagonists. In contrast, we expected po-

sitive interactions based on the hypothesis that the polymers could

interact by non-ionic side chains and by electrostatic interactions

Table 3

Coefficients of the polynomial models for the response variables

Factors EAI (nm) D[4,3] (lm) CI (%)

PEC (b1) 0.29 ± 0.01 9.3 ± 0.4 87.0 ± 1.6

TSP (b2) 0.15 ± 0.01 17.9 ± 0.4 69.7 ± 1.3

SPI (b3) 0.14 ± 0.01 20.2 ± 0.4 59.4 ± 1.3

PEC TSP (b12) 0.12 ± 0.03 8.8 ± 1.6 55.3± 6.1

PEC SPI (b13) 0.20 ± 0.03 34.0 ± 1.8 165.0± 6.1

TSP SPI (b23) 0.07 ± 0.03 23.3 ± 1.7 142.4± 5.8PEC + TSP + SPI (b123) ns ns ns

p (model) <0.01 <0.01 <0.01

DF (model/total error) 5/33 5/59 5/27

p (lack of fit/pure error) 0.68 0.31 0.08

Adjusted R2 0.86 0.95 0.98

Table 4

Observed and estimated values obtained for the three additional points inserted in the experimental design a

Additional points PEC ( x1) TSP ( x2) SPI ( x3) EAI (nm) D[4,3] (lm) CI (%)

PEC + TSP + SPIb 0.67 0.17 0.17 0.199 (0.192 0.206) 13.6 (11.5 –15.7) 61.1 (57.8–64.3)

PEC + TSP + SPIc 0.67 0.17 0.17 0.203 (0.194 0.212) 18.1 (17.6 –18.5) 50.5 (48.9 –52.2)

PEC + TSP + SPIb 0.17 0.67 0.17 0.144 (0.139–0.149) 19.5 (17.8 –21.1) 32.8 (28.7 –36.9)

PEC + TSP + SPIc 0.17 0.67 0.17 0.145 (0.137–0.153) 21.4 (21.0 –21.8) 43.9 (42.4 –45.5)

PEC + TSP + SPIb 0.17 0.17 0.67 0.132 (0.127–0.138) 24.2 (23.6–24.8) 30.0 (30.0– 30.0)

PEC + TSP + SPI*c 0.17 0.17 0.67 0.133 (0.124 –0.141) 24.7 (24.2–25.1) 29.8 (28.2–31.4)

v2 (probability values)d – – – p = 0.99 p = 0.52 p = 0.08

a Values are expressed as mean (±95%) confident interval.b Experimentally observed values.c

Values estimated by the respective models.d Probability obtained by Chi-Square test comparing observed and predicted values.

Table 5

Chemical composition and emulsifying properties of the mixtures containing soy

proteins and pectin before (natural) and after extrusion (extruded)

Nutrients (g/100g)a Mixtures containing soy protein and pectin

Natural Extruded pd

Moisture 7.7 ± 0.1 11.4 ± 0.2 <0.01

Proteinb 41.1 ± 0.9 41.4 ± 0.7 0.85

Lipids 0.9 ± 0.1 0.4 ± 0.2 0.23

Ashes 4.6 ± 0.1 4.3 ± 0.2 0.36

Carbohydratec 21.9 19.5 –

Insoluble fiber 6.8 ± 0.6 5.9 ± 0.9 0.07

Soluble fiber 17.0 ± 1.3 17.1 ± 2.2 0.93

EAI (nm) 0.273 ± 0.008 0.385 ± 0.019 <0.01

CI (%) 36.1 ± 3.0 33.4 ± 3.1 0.41

a Values are mean± SD (n = 3).b Calculated by using N 6.25 for all samples.c Values obtained by difference.d Probability value obtained by T -test for dependent samples.




involving the positive amino acids of the protein and the negative

acid groups of the pectin, as exemplified by Benichou et al. (2002)

to proteins and carrageenans.

Some authors have reported synergism in theemulsifyingability

when proteins are mixed with polysaccharides. Uruakpa and Arnt-

field (2005) observed that isolated canola protein combined with k-

carrageenan increased emulsifying activity and stability, due to the

electrostatic complex formation and enhanced molecular interac-

tion at pH 6.0. Chain association, which results in network forma-

tion, is believed to also be due to the interaction between theester and hydroxyl groups (Williams et al., 2005). Tolstoguzov

(1991) stated that the addition of an equal weight of pectin to a

solution of legumin increased the emulsion stability under condi-

tions of incompatibility (pH 7.6). According to this study, the use

of a polysaccharide (pectin) incompatible with the protein(legumin

in soybean for example) intensifies the protein adsorption and de-

creases the requirements for protein content sufficient for a multi-

layer adsorption. Neirynck et al. (2007) postulated that pectin had a

stabilizing effect on the protein-stabilized emulsion above the pro-

tein isoelectric point due to the combined electrostatic and steric

repulsion effects at the interface. Due to these previous results,

we expected to find synergistic behaviour between soybean pro-

teins and pectin in our study. However, contour curves (Fig. 1)

showed that no synergism was observed when pectin was mixedwith the two forms of soybean protein, isolated and textured.

In neutral solutions, pectin, even when it contains a high meth-

oxylation degree, is negatively charged. Thus, a net repulsion prob-

ably occurred among the polymers at pH levels higher than the

isolelectric point of the protein, since both carried the same electric

charge. The importance of the pH in the intramolecular association

between incompatible polysaccharides and proteins is clear (Ein-

horn-Stoll et al., 2005; Liu et al., 2008). The pH (6.2) adopted in

our study was slightly above the isoeletric point of the isolated

soybean protein (4–5). At this pH value, the isolated soybean pro-

tein predominantly shows weak negative electrical charge, becom-ing partially soluble (<35%), while the textured soybean protein is

precipitated in function of the higher proportion of hydrophobic

residues exposed by denaturation (Fig. 3). The minimum solubility

requirement for a good emulsifying performance may vary among

proteins (Damodaran, 1996). Therefore, the low solubility of the

soybean proteins used in our study could have contributed to the

negative interaction with pectin, observed through the polynomial

binary coefficients signals (Table 3).

The mixture of pectin with isolated soybean protein (b12), pro-

duced worse results than a mixture of pectin and textured soybean

protein (b13). The partial heat denaturation of proteins can improve

their surface activity and emulsifying properties (Damodaran,

2005). Soybean protein isolates, in a natural or denatured state,

displayed different behaviour as emulsifying agents (Scuriattiet al., 2003). The heating of proteins may result in changes of struc-

0.28

0.26

0.24

0.22

0.20

0.18

0.16

0.14

0.00

0.25

0.50

0.75

1.00

SPI

0.00

0.25

0.50

0.75

1.00

PEC

0.00 0.25 0.50 0.75 1.00

TSP

EAI (nm)

24

22

20

18

16

14

12

10

0.00

0.25

0.50

0.75

1.00

SPI

0.00

0.25

0.50

0.75

1.00

PEC

0.00 0.25 0.50 0.75 1.00

TSP

D[4.3]

( m)

80

70

60

50

4030

0.00

0.25

0.50

0.75

1.00

SPI

0.00

0.25

0.50

0.75

1.00

PEC

0.00 0.25 0.50 0.75 1.00

PTS

CI (%)

a b

c

Fig. 1. Contour plots showing the effects of pectin (PEC), textured soybean protein (TSP) and isolated soybean protein (SPI) proportion on EAI (a), D[4,3] (b) and CI (c). Each

contour line corresponds to a given response value.




ture and functional properties such as an unfolding of the mole-

cules and exposing functional groups, such as cationic and hydro-

phobic groups, from the inside (Tolstoguzov, 1991; Benichou et al.,

2002; Einhorn-Stoll et al., 2005). The effect of extrusion on soybean

proteins solubility can be clearly observed in Fig. 3, where the

higher exposition of hydrophobic groups promoted an expressive

drop in solubility of the textured soybean protein. Although low

solubility is a negative factor for the emulsifying properties of pro-

teins, we think that this fact was positive in our study, contributing

to a reduction in the electrostatic repulsion between pectin and

soybean proteins. This effect might have been responsible for the

better interaction between textured soybean protein and pectinthan isolated soybean protein and pectin.

Although the extrusion process promotes an increase in the sol-

uble fiber followed by a decrease in the insoluble fiber (Larrea

et al., 2005), no significant changes were observed in our study

in the fiber content of the mixtures before and after extrusion.

Our data also suggest that the denaturation caused by the extru-sion of the soybean protein and the proteinaceus fraction of pectin

may have promoted a change in the hydrophobic and hydrophilic

amino acid distribution at the molecule surface and, consequently,

improved the interfacial properties, as discussed previously. The

mixture submitted to extrusion showed an emulsifying capacity

41% higher than the non-extruded mixture.

5. Conclusion

The results of our study showed that it is possible to estimate

the emulsifying capacity and stability of mixtures containing two

types of soybean proteins and pectin by response surface method-

ology. Taking into account that soybean proteins are extensively

applied as emulsifiers in processed food products, our resultsshowed that pectin could substitute or improve the emulsifying

properties of the soybean proteins, and that the extrusion

brings additional advantage to interfacial properties of this

combination.

Acknowledgement

Financial support from the FAPESP (Process 06/00384-0 and 07/

02682-0) is acknowledged.

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Soybean Proteins by Response Surface