Food Hydrocolloids 19 (2005) 595604 www.elsevier.

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Coalescence and occulation in o/w emulsions of native and denatured whey soy proteins in comparison with soy protein isolates
Gonzalo G. Palazoloa, Delia A. Sorgentinib, Jorge R. Wagnera,b,*
n y Desarrollo en Criotecnolog a de Alimentos (CIDCA), CONICET, Facultad de Ciencias Exactas, Centro de Investigacio Universidad Nacional de La Plata, Calle 47 y 116, 1900 La Plata, Argentina b gicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115, 1900 La Plata, Argentina Departamento de Ciencias Biolo
a

Abstract Comparison of coalescence and occulation stability of o/w emulsions prepared with whey soy protein and soy protein isolates, native and denatured (NWSP, NSI; DWSP, DSI, respectively) was performed. Sodium caseinate (SC) was used as emulsier control. Emulsions, without and with NaCl (500 mM in aqueous phase) were analysed by backscattering during 24 h in quiescent storage. In the absence of salt, the cream phase global destabilization percentage (D%) at 24 h of NWSP emulsions was the highest, whereas in the presence of NaCl not only NWSP but also NSI emulsions were destabilised. Cream phase stability, expressed as coalescence (C%) and coalescence plus occulation percentages (CCF%) was calculated from mean droplet size (D43) without and with 1% SDS. C% and CCF% had a polynomial correlation with D%, which indicate that this parameter could reect the particle size increase due to both coalescence and occulation processes. The higher stability of denatured soy protein (DWSP, DSI) emulsions respect to those prepared with native proteins could be related with the presence of hydrated ocs formed during quiescent storage. In these emulsions, high elastic modulus (G 0 ) and low oil volume fraction (f) values indicated an hydrated and gel-like structure of cream phase. Thermal denatured whey soy proteins, without antitryptic activity, allowed to obtain as coalescence stable emulsions as those prepared with SC. Although backscattering method detected changes in particle size with a less sensibility than droplet size determination, it allowed the continue evaluation of global stability in non-diluted emulsions. q 2005 Elsevier Ltd. All rights reserved.
Keywords: Backscattering; Coalescence and occulation stability; Cream phase; o/w Emulsions; Whey soy proteins

1. Introduction Coalescence stability affects processing or shelf life of oil in water emulsions. Depending of product high or low coalescence stability is desired (Britten & Giroux, 1991). The coalescence probability increases as the uctuations in the membrane shape may them become large enough to form a hole, which extends from one droplet to another. The magnitude of the shape uctuations is governed by the interfacial tension, lm rheology and mechanical applied forces (Evans & Wennerstrom, 1994; McClements, 1999).
n y Desarrollo * Corresponding author. Address: Centro de Investigacio a de Alimentos (CIDCA), CONICET, Facultad de en Criotecnolog Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 116, 1900 La Plata, Argentina. Tel./fax: C54 221 4254853/4249287/4890741. E-mail address: jrwagner@biol.unlp.edu.ar (J.R. Wagner). 0268-005X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2004.10.022

The emulsifying properties of isolate soybean proteins, which contains the storage proteins, glycinin and b-conglycinin, have been studied (Kinsella, 1979; Peng, Quass, Dayton, & Allen, 1984). However, whey soy proteins need further research. The main components of this fraction are the Kunitz trypsin inhibitor and lectins with a molecular mass of 20 and 120 kDa, respectively. The antinutritional activity and thermolability of these proteins have been reported in previous works (Liener, 1981). In previous works in our laboratory, structural characteristics and surface behaviour of native and thermal denatured whey and storage soy proteins were studied (Palazolo, Sorgentini, & Wagner, 2004; Sorgentini & Wagner, 1999). The coalescence of o/w emulsions prepared with proteins is a process strongly accelerated in shear stress conditions because it is slow in comparison with creaming and occulation (Britten & Giroux, 1991). Mitidieri and

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Wagner (2002) analysed the inuence of protein denaturation and salt addition on coalescence stability of oil in water emulsions formulated with whey soy proteins under stirring in controlled conditions. Instead, in this work, the coalescence destabilization of emulsion was studied under quiescent conditions using a vertical scan analyser. The physical evolution of this process is followed without disturbing the original system and with good accuracy and s, & An n, 2002). Furthermore, o reproducibility (Pan, Toma the inuence on coalescence stability of native or denatured whey soy proteins and of a very common process in food industry such as salt addition was studied. This inuence was analysed in comparison with two protein ingredients fully utilised in food industry: soy protein isolates and sodium caseinate.

S25N10G device, IKA Labortechnik, Karlsruhe, Germany). Assays were performed at least twice. 2.4. Global destabilization of emulsions Destabilization was estimated using a vertical scan analyser (Quick Scan, Beckman Coulter). The samples were put in a cylindrical glass measurement cell and the backscattering (BS) and transmission (T) proles as a function of the sample height (total heightZ60 mm) were studied in quiescent conditions at 20 8C during 24 h. Destabilisation kinetic of cream phase were followed by measuring the mean values of BS (BSav in 10 mm of middle zone of cream phase, named B zone, see Fig. 1) as a function of time. Global destabilisation percentage (D%) was estimated from the expression:   BSav max K BSav t D t Z ! 100 (1) BSav max

2. Materials and methods 2.1. Materials Defatted solvent-free soy our, prepared under controlled conditions (not thermally inactived to avoid protein denaturation), was provided by Santista S.A., Brazil. The sodium caseinate was purchased by Sigma Co. and was utilised without further purication. All the other reagents were analytic grade. 2.2. Preparation of native soy isolate (NSI), native whey soy proteins (NWSP), and denatured fractions (DSI, DWSP) Native soy isolates (NSI) was prepared by isoelectric precipitation (pH 4.5) of pH 8.0 aqueous extract of deffated soy our. From the supernatant of this precipitation, native whey soy proteins (NWSP) was obtained by salting out (90% saturation ammonium sulphate). Details were described in previous works (Mitidieri & Wagner, 2002; Sorgentini & Wagner, 1999). The protein content in NSI was 90G0.1% w/w (N!6.25) and protein content in NWSP was 99G0.5% w/w (N!6.25). Total denaturation of both fractions was achieved by heating the respective solutions (1 mg/ml, 10 mM sodium phosphate, pH 7.0) to 90 8C for 5 min with further cooling in a waterbath at 20 8C. These fractions (DSI and DWSP) were used immediately after their preparation. 2.3. Preparation of o/w emulsions NSI, NWSP, DSI, DWSP and SC were dispersed in 10 mM phosphate buffer pH 7.0 (without and with 500 mM NaCl in the aqueous phase) at nal concentration of 0.1% (1 mg protein sample/ml buffer). The emulsions were prepared with 10 ml of each solution and 5 ml of commercial rened sunower oil by homogenisation at 20,000 rpm for 60 s (Ultraturrax T-25 dispersing tool,

Fig. 1. BS% proles corresponding to emulsions prepared with: (a) NWSP, (b) NSI, and (c) SC. The arrows position indicate the middle zone of band B. Tube length: 60 mm, temperature: 20 8C.

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where BSav max is the maximum mean value of backscattering corresponding to the middle zone of creamed phase and BSav t is the mean value of backscattering at quiescent storage time t. Determinations were conducted at least in duplicate. 2.5. Determination of droplet size distribution The droplet size distribution were determined in the range 0.03360 mm by laser scattering using a Micro Particle Analyser (Malvern Mastersizer 2S, Malvern Instruments Ltd, UK). Immediately after the preparation of emulsions, two aliquots were diluted (1:1 v/v) with 10 mM sodium phosphate buffer pH 7.0 or 50 mM Tris/HCl buffer pH 8.0 containing 1.0% SDS. Measurement in the presence of SDS allowed to evaluate the individual droplet size without occulation (Anton, Beaumal, Brossard, Llamas, & Le Denmat, 2002; McClements, 1999). The adequate presentation (4OHD presentation) was selected to take into account the refractive index of the oil and the adsorption caused by interfacial lm. The size of 90% of droplet in all emulsions was !1.5 mm when the distribution was expressed as particle number. D43 values, obtained from the droplet size distribution expressed as oil volume percentage containing in each range of droplet size, were used to calculate coalescence ( C %), coalescence plus occulation (( CC F)%) and occulation (F%) percentages during 24 h in quiescent conditions, using the following expressions: C % Z D43CSDS K D43 i =D43 i ! 100 C C F % Z D43KSDS K D43 i =D43 i ! 100 F % Z C C F % K C % (2) (3) (4)

parallel-plate sensor. Aliquots of cream layer was on the lower plate, which was maintained at 20 8C. The equipment was driven through the Haake software osc.2.0. Experimental data were obtained by recording the storage (G 0 ), loss (G 00 ) and tan d (G 00 /G 0 ) as a function of oscillation frequency (0.0329.6 Hz range) within the linear viscoelasticity range (strainZ3%). Tests were conducted at least in triplicate. 2.8. Statistical analysis Data were analysed by analysis of variance and signicance difference between the Fishers test (Systat version 5.0). An alpha level of 0.05 (p!0.05) was used to determine signicance.

3. Results and discussion 3.1. Global destabilization of o/w emulsions When o/w emulsions are prepared with proteins as the only emulsifying agent, in low concentrations and under quiescent conditions, coalescence becomes a slow destabilising mechanism, compared to creaming and occulation (Britten & Giroux, 1991). On this basis, emulsions were stored under quiescent conditions at room temperature (20 8C) for 24 h, enough time to detect coalescence. Fig. 1 shows backscattering (BS) proles of emulsions prepared with native whey soy proteins (NWSP), native soy isolates (NSI) and sodium caseinate (SC) (Fig. 1ac, respectively). Zone I indicates creaming destabilisation and zone II is the cream phase that remained after oil droplets have accumulated in the upper part of the tube. To compare cream phases of different emulsions, the BS proles vs. tube height of Fig. 1 was divided in three zones: A, B and C, corresponding to low, medium and high parts of the cream phase, respectively. SC emulsion prole (Fig. 1c, control) shows a peak at 60 min. previous to zone A, which was attributed to the accumulation of small droplets with low creaming velocity. Besides, zone C shows another marked peak that should indicate the presence of foam in emulsion. To analyse coalescence process on cream phase, it is necessary to minimise the incidence of the processes previously mentioned: accumulation of tiny droplets and of air bubbles. Thus, in the middle zone (zone B) of cream phase, a drop on BS prole can be seen as a decrease in the number of droplets, what is, in turn, mainly generated by either a coalescence alone or coalescence plus a occulation process. Even though droplet migration due to creaming may exist, this process should be minimised by the high viscosity of cream phase (Mc Clements, 1999). For NWSP emulsion, zone B showed a marked decrease on 24 h-BS prole compared to the prole obtained during the rst hour of destabilization (Fig. 1a). However, emulsions containing NSI and SC did not show this decrease. These results may indicate, that in NWSP emulsion a marked increase of

where D43 i is the mean droplet size of initial emulsion; D43KSDS and D43CSDS are the mean droplet sizes of the middle zone of cream phase measured at 24 h in the absence and the presence of SDS, respectively. In addition, occulation degree in cream phase at 24 h (FD%) were calculated as: FD% Z D43KSDS K D43CSDS =D43CSDS ! 100 Determinations were conducted at least twice. 2.6. Oil volume fraction of cream phase Emulsions were stored for 24 h in quiescent conditions. The cream layer was carefully separated from serum and oil volume fraction (f) was estimated according to the method reported by Hill (1996). Assays were conducted at least in duplicate. 2.7. Rheological behaviour of cream phases Tests were conducted in a Haake CV20 rheometer (Haake, Karlsruhe, Germany) with a NV rotor using a 1 mm-gap (5)

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droplet size may have been produced at 24 h. On the other hand, an increment of BS in the bottom part of tube (zone I) at 24 h was observed, specially to NWSP emulsion (Fig. 1a). This was not attributed to sedimentation process because in that zone an concomitant increment of T was also observed. At an tube height of 10 mm, the T values were of 42, 12 and 8% for NWSP, NSI and SC emulsions, respectively. To analyse destabilization kinetics, variation of BSav values of zone B as a function of time obtained from the BS proles of all assayed emulsion were recorded (Fig. 2af). All proles showed an initial increase of BSav up to 30 min. This could be attributed to an increase of the number of oil droplets due to the formation of the cream phase at different times depending on creaming velocity. The time needed for BSav to reach its maximum value (BSav max) increased with increasing global stability of the emulsion. Particularly, NWSP emulsion reached BSav max at 3 min. (Fig. 2a), because at that time the rate of droplet number diminution was higher than droplets accumulation. After this time, a decrease of BSav up to 24 h was observed. This decrease was not attributed to a reduction of particle number by a sedimentation process, as it was mentioned above. For whey soy proteins thermally denatured (DWSP), BSav max shifted to higher values (18 min) and BSav remained constant up to 60 min, with a slight decrease at 24 h (BSav 24), indicating a high global stability (Fig. 2a). On the contrary to NWSP, native soy isolate (NSI) gave a very stable emulsion, with BSav max constant between 20 min and 24 h (Fig. 2b). Emulsion with denatured isolate (DSI) did not show stability differences with regard to NSI. However it showed a shift of BSav max to higher values (z40 min) due to its great creaming stability. SC emulsion showed a different behaviour compared to soy proteins; BSav max value was reached at 50 min, indicating a slow rate of stable cream phase formation (Fig. 2c). The effect of 500 mM NaCl on the stability of the cream phase of the emulsions can be evaluated by comparing Fig. 2df (emulsions with NaCl) with Fig. 2ac (emulsions without NaCl). Emulsions of NWSP and NSI were destabilised by salt presence (mainly the rst one) as seen by a reduction of BSav 24. However, the stability of emulsions with denatured proteins (DWSP and DSI) remained almost unaltered with the increase of ionic strength. Improvement of surface hydrophobicity and presence of low molecular mass species as a consequence of thermal treatment would explain the stability of interfacial lm formed by denatured soy proteins (Mitidieri & Wagner, 2002; Palazolo et al., 2004). In general, NaCl presence in these emulsions led to a decrease of BSav along the whole assayed period and a shift of BSav max towards shorter times (compare Fig. 2d and e with a and b). Both changes could be associated with an increase of creaming process due to the higher initial particle size induced by NaCl (Palazolo et al., 2004). For SC emulsion, salt presence slightly decreased BSav value while BSav max was not modied (compared Fig. 2f with c).

D% was obtained from BS proles and calculated from Eq. (1) (Fig. 2af). This parameter allowed to quantify global destabilization of cream phase from its formation to the end of storage (24 h). Since it is an index of BSav decrease associated to a lower number of particles, it can be related with coalescence and/or occulation. Table 1 shows D% values at 24 h for all assayed formulations. NWSP emulsion exhibited the highest destabilization degree. Stability increased with denatured protein presence, as indicated by the low D% values of DWSP and DSI emulsions, that behaved similarly to those prepared with SC. Addition of NaCl had a negative effect on stability of NWSP and NSI emulsions, whereas D% did not change signicantly in emulsions prepared with denatured proteins and SC (p!0.05). The D% values calculated at tZ60 min (data not shown) were lower than those at 24 h but they exhibited the same tendency. 3.2. Relationship between cream phase destabilization and rheology During migration process droplets ascend to the upper part of the tube, droplets collide between each other, and during that period stability against coalescence is mainly governed by the interfacial lm resistance. Total collision frequency between droplets (FT) of both, a just prepared or stored under quiescent conditions emulsions can be considered as the contribution of two factors, one attributed to the Brownian movement and the other to the movement of the droplets under a gravitational eld. Once cream phase is already formed, FT is minimum and coalescence process is mainly governed by shape uctuations and interfacial lm movement due to a long contact between oil droplets. Coalescence probability increases when uctuations become so important to form holes that can pass from one droplet to another. Under these conditions, emulsions may become stable when the thickness of the continuous phase around the droplets (interstitial continuous phase) is enough to avoid contact between droplet lms, and when interfacial uctuations do not lead to the exclusion of interstitial water (Mc Clements, 1999). Thus, a lower volumetric fraction of oil (f) and consequently a higher interstitial aqueous phase within the cream phase, may minimise coalescence. Other important factors that directly affect coalescence stability are the rheological behaviour of the interfacial lm and the global rheology of cream phase, which is mainly controlled by the presence of ocs. On these basis, BSav vs. time proles (Fig. 2) and D% values (Table 1) were analysed. Interfacial lm weakness of NWSP emulsions was evidenced under stirring, as reported in a previous work (Mitidieri & Wagner, 2002). NWSP are mainly constituted by the Kunitz antitryptic factor and lectin, which are unable to give a resistant lm due to their low molecular weight and low surface hydrophobicity. In the present work, even though emulsions do not suffer shear stress, NWSP emulsion showed a low stability as well (Fig. 1a). During the quick

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Fig. 2. Destabilization kinetics (BSav as a function of time) for emulsions prepared with various protein samples. In the absence of NaCl: (a) () NWSP (B) DWSP, (b) (&) NSI (,) DSI y, (c) (:) SC. With salt addition (500 mM in the aqueous phase, before homogenisation): (d) () NWSP (B) DWSP, (e) (&) NSI (,) DSI y, (f) (:) SC. Temperature: 20 8C.

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Table 1 Stability of NWSP, DWSP, NSI, DSI, and SC emulsions and oil volume fraction (f) corresponding to their 24 h cream phases Sample NWSP DWSP NSI DSI SC NaCl 0 0.5 M 0 0.5 M 0 0.5 M 0 0.5 M 0 0.5 M D (%) 27.3G2.9 48.1G4.2 2.4G0.2 0.1G(!0.1) 1.7G(!0.1) 18.2G2.7 0.4G(!0.1) 0.1G(!0.1) 0.1G(!0.1) 0.00 f (dimensionless) 0.788G0.002 0.917G0.016 0.611G0.001 0.637G0.002 0.734G0.009 0.844G0.014 0.668G0.004 0.649G0.005 0.698G0.004 0.707G0.004

a very low D% (!3, Table 1). The high hydration degree of the cream phase of these emulsions, higher for DWSP than for DSI and SC, was demonstrated by the low values of f, which did not markedly change with NaCl addition. Besides, rheological analysis of the cream phase without salt showed that DWSP emulsion had the highest G 0 value (oscillation frequencyZ1 Hz, Fig. 3a). DSI, SC and NSI

D% was the global destabilisation estimated from BS proles. LSD (p!0.05) was 4.4 and 0.011 for D% and f, respectively.

formation of the cream phase of this emulsion, droplets moved and collided freely. Since BSav decreased rapid and immediately after reaching BSav max (Fig. 2a), coalescence process of NWSP emulsion should have started together with creaming. This behaviour can be attributed to the mentioned lm weakness formed by native proteins of soy whey. At 24 h storage, cream phase showed a low hydration degree (high f), what favoured destabilization (D%Z27.3, Table 1). With addition of NaCl, the destabilization was more marked (Fig. 2d), due to the efciency increase of collisions because of the electrostatic interactions screening on interfacial lm. After 24 h, this fact was evidenced with a great increase of f (O0.9), and consequently, a high value of D% (48.1, Table 1). In the case of NSI emulsions, the formation of a resistant lm avoided coalescence destabilization. It is well known that native 7S (b-conglycinin) and 11S (glycinin) globulins, even at low concentrations, form a viscoelastic resistant lm at oil/water interface. The lm is characterised by a high structuration degree; it is also favoured by the high molecular mass of the proteins (270 and 360 kDa, respectively) and its relatively high surface hydrophobicity (Mitidieri & Wagner, 2002). In the cream phase, these resistance and structuration of the lm would help to avoid droplet deformation. Besides, electrostatic and steric repulsions between adsorbed polypeptidic chains would also avoid droplet approaching, maintaining interstitial aqueous phase thickness. Thus, the combination of all these effects would explain the relative stability of these emulsions. On the other hand, NaCl presence would favour the screening of electrostatic interactions and the consequent hydrophobic interaction increase, what would promote approaching between droplets and a higher probability of interlm interaction. These facts are evidenced by an increase of f (lower hydration of cream phase) and a higher destabilization degree at 24 h (D%Z18.2, Table 1). Emulsions with denatured proteins, DWSP and DSI, were more stable to coalescence regardless the ionic strength, similarly to SC emulsions (Table 1). BSav 24 values of cream phase were above 58% (Fig. 2), giving

Fig. 3. Elastic (G 0 ) and viscous (G 00 ) behaviour of cream phases at 24 h for NWSP, DWSP, NSI, DSI and SC emulsions: (a) without NaCl, (b) in the presence of salt (500 mM in the aqueous phase before homogenisation), (c) tan dZtan (G 00 /G 0 ). Oscillation frequency: 1 Hz. Strain: 3%, in the linear viscoelastic range.

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emulsions gave similar and lower G 0 values, while NWSP emulsion gave the lowest one (p!0.05). The same tendency was observed along entire oscillation frequency range (data not shown). According to these observations, a relationship between G 0 values and hydration degree of cream phase should exist; the higher G 0 , the lower f (Table 1). Effectively, in the presence of salt, a relationship between G 0 and cream hydration was observed, as well (Fig. 3b). DWSP and DSI emulsions, with high G 0 values showed the more hydrated creams, while NWSP emulsion with a minimum G 0 had a low hydrated cream (fZ0.92) (Table 1). In all emulsions, the elastic component (G 0 ) predominated over the viscous one (G 00 ), especially for DWSP with and without salt (G 0 almost four times higher than G 00 without salt and approximately nine times at high ionic forces) and for DSI with salt (Fig. 3a and b). Thus, the relationship between both moduli (tan dZG 00 /G 0 ) was below one for all the creams (Fig. 3c) indicating a gel type behaviour, which was evident for DWSP. The tan d values decreased with salt presence, what could be attributed to a higher cream structuration. The only emulsion markedly destabilised by coalescence under the absence of NaCl was that with NWSP, which showed the lowest G 0 and G 00 values for the cream phase. This fact could be related with both its lower hydration degree and its interfacial lm weakness. As mentioned previously, NWSP and NSI emulsions with NaCl markedly coalesce under quiescent storage (Table 1), what was expected according to the low G 0 and G 00 values (Fig. 3b). An increase of both moduli was observed for cream phases of SC emulsion, characterised by low coalescence degrees (Fig. 3b). Thus, its stability cannot be attributed to the gel type structure but to the interfacial lm resistance. However, DWSP and DSI emulsion with NaCl showed a marked increase of G 0 corresponding to a tan d decrease (Fig. 3c), indicating that the gel type structure of cream phase granted the high stability against coalescence.

3.3. Relationship between destabilization analysed by Quick Scan and droplet size distribution To analyse if destabilization measured by Quick Scan can be attributed to changes of droplet size and/or oc presence, mean diameters D43 of initial emulsions and of the cream phase (zone B) were measured, both with and without SDS (Table 2). The D43 parameter allows to detect coalescence and occulation process with more sensibility. Dilution and stirring are likely to disrupt any weakly occulated droplets but leave strongly occulated droplets intact. In occulated emulsions the droplets aggregate in heterogeneous particles which have an ill-dened refractive index and shape. Consequently the particle size distribution determined by light scattering gives only an approximate indication of the true size of the ocs. However, even if the D43 values could be absolutely reliable, a large increase in D43 reects the association of the emulsion droplets into large ocs. Such a method was largely used in view to compare occulation degree of different emulsions placed in similar conditions (Anton et al., 2002). The D43 increase by effect of NaCl addition in initial emulsions and by effect of quiescent storage at 24 h was showed in Table 2. In the absence of NaCl but most noticeable in its presence, emulsions prepared with native proteins showed a higher trend to suffer destabilization than denatured proteins and SC. Both simultaneous processes, coalescence and occulation occur as was reected by high values of (CCF)% and C%. Fig. 4 shows the droplet size distribution of emulsions prepared with soy protein samples, with 500 mM NaCl, which correspond to the greatest destabilization condition. For emulsions prepared with native proteins (NWSP and NSI), cream phase distributions at 24 h shifted towards larger sizes respect to initial emulsions (Fig. 4a and c). Moreover, the distributions for NWSP cream phase in the absence and presence of 1% SDS were different, indicating the simultaneous destabilization by coalescence and occulation (Fig. 4a). The major peak (D43O100 mm) observed

Table 2 Particle size of initial emulsions and cream phases (24 h, zone B) for NWSP, DWSP, NSI, DSI and SC emulsions Emulsion NaCl Initial D43 (mm) 43.0 43.3 44.7 47.9 39.7 59.1 44.0 52.2 52.3 41.7 D43 (cream phase, 24 h) Without SDS (mm) 81.1 45.8 56.8 50.5 45.3 119.1 46.7 85.9 51.6 45.4 With SDS (mm) 66.7 44.9 53.5 50.2 44.0 106.2 43.9 79.3 52.3 44.5 88.6 5.7 27.0 5.5 14.3 101.7 6.1 64.5 0 8.9 55.3 3.8 19.8 5.0 10.8 79.8 0 51.8 0 6.6 33.3 1.9 7.2 0.5 3.5 21.9 6.1 12.7 0 2.3 21.5 1.8 6.0 0.7 3.1 12.1 6.3 8.3 0 2.1 (CCF)% C% F% FD%

NWSP DWSP NSI DSI SC NWSP DWSP NSI DSI SC

0.5 M

Coalescence (C%), coalescence plus occulation (CCF%), occulation (F%) percentages and cream phase occulation degree (FD%, quiescent storage at 24 h) for these emulsions. Maximum standard deviation: 1%.

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Fig. 4. Droplet size distribution of initial emulsions and cream phases after quiescent storage (24 h), with 500 mM NaCl addition (in the aqueous phase). Measurements were carried out in the absence and in the presence of SDS (1%, Tris/HCl buffer, pH 8.0): (a) NWSP, (b) DWSP, (c) NSI, and (d) DSI.

without SDS, could be attributed to contribution of ocs and coalesced droplets, because it was split in two peaks with SDS. In turn, the coalescence was the main mechanism of cream phase destabilization of NSI emulsions, as evidenced by a similar droplet size distribution with or without SDS (Fig. 4c). On the other hand, the high cream phase stability of emulsions prepared with DWSP and DSI was showed in Fig. 4b and d, indicating a correspondence with very low (CCF)% and C% values (Table 2). Fig. 5 shows that (CCF)% and C% exhibit a good polynomial correlation with the global destabilization, D%, obtained from Quick Scan proles. It can be observed that the D% increase correspond to the increase of particle size due to both coalescence and occulation processes. However, the vertical scan analyser detect changes in the particle size with a less sensibility than those recorded with

the particle analyser. D% was not measurable for (CCF)% !20, and it is an underestimation of changes in particle size (D%!(CCF)% for all emulsions), probably due to the different measurement conditions. Notwithstanding this, the analysis of BS proles as a function of time offers the advantage to allow a continue evaluation of the emulsion global destabilization without previous dilution (Fig. 2). The negative effect of NaCl on the cream phase stability of NSI and NWSP emulsions could be explained taking into account the cream phase characteristics. Flocculation percentage (F%) is indicative of occulation destabilization of emulsion during storage time total period (024 h). According to Table 2, the main effect of NaCl was to enhance the C% values as a consequence of the diminution in the ocs amount (F%) by large droplets formation. On the other hand, cream phase occulation degree at 24 h (FD%)

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found. Those with low hydration degree due to a high interaction between lms with exclusion of interstitial water, showing a high shear stability during the particle size measurement. The free mobility of these highly compact ocs (low hydrodynamic volume) would explain the low viscoelastic behaviour of the cream phases of NWSP and NSI emulsions. On the other hand, those ocs with high hydration degree do not resist extreme turbulence and high dilution condition because the droplets were held together by secondary minimum forces. These ocs, formed by the interaction of hydrated lms that give a gel type structure, are mainly present in DWSP emulsions. Then, thermal denaturation of soy whey proteins, besides to allow their use in human foods by antinutritional factor inactivation, could confer the ability to form coalescence resistant emulsions during storage.
Fig. 5. Relationship between C%, (CCF)% and D% for all emulsions. C% and (CCF)% were obtained from droplet size distributions; D% was obtained from backscattering (BS%) proles.

Acknowledgements The author acknowledges the nancial support from the n Cient ca y Tecnolo gica Agencia Nacional de Promocio (FONCyT, PICT 98-09-04296) and the Universidad Nacional de La Plata, Argentina (Project 11/X279). Authors wish as de Ma z S.A.I.C.F, Planta Hellmanns. to thank Rener (Unilever Bestfoods Argentina, Parque Industrial Pilar, Provincia de Buenos Aires) for kindly providing us the Malvern Mastersizer Micro Particle Analyser. J. R. Wagner is member of Consejo Nacional de Investigaciones cas y Te cnicas (CONICET) and G. G. Palazolo is Cient n de Investigaciones Cient cas research fellow of Comisio de la Provincia de Buenos Aires (CIC).

for native proteins was higher than those of denatured proteins and SC (Table 2). The formed ocs would resist the effect of turbulence and high dilution conditions during the measurement of particle size. This suggests a bridging occulation mechanism, showing that interaction between droplet lms was not mediated by an interstitial aqueous phase (McClements, 1999). This result is in accordance with the cream phase low hydration degree of NWSP and NSI emulsions (Table 1). Table 2 shows that F% was higher than FD% for emulsions prepared with NSI and NWSP which indicate, as previously mentioned, that some formed ocs coalesced in this condition. Besides, cream phase of DSI and DWSP emulsions exhibited a high hydration degree (Table 1) which could be explain by open structure ocs, in which droplets are held together by weak interactions (Palazolo et al., 2004). Since these hydrated ocs were not so stable to the turbulence and high dilution generated during particle size measurement, a low FD% was obtained (!10, Table 2). For these emulsions, F% and FD% values were similar (Table 2), indicating that formed ocs did not coalesce during the storage total time period due to the high amount of intersticial aqueous phase (low f value, Table 1).

References
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4. Conclusion The global destabilization measured with backscattering method was attributed to both simultaneous processes, coalescence and occulation, which can be differentiate with the particle analyser. The higher stability of denatured soy protein emulsions respect to those prepared with native proteins could be explained by the nature of the interfacial lms and ocs formed during quiescent storage. Then, two types of ocs that differ in their hydration degree were

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