s:su

UNI

MicrostructureEmulsionRheologyStabilityHigh-pressure homogenization

ofof p

cible l

speed (1000e25,000 rpm) in a narrow gap (50e1000 mm) betweena rotating disk (rotor) and a static disk (stator). This processpromotes the stretch and break-up of the particles of the dispersedphase by mechanical impingement against the wall, due to highuid acceleration and shear stress in the turbulent ow in the gap.Droplet mean with diameters smaller than 1 mm cannot be

reducing the surface tension, protecting the oil droplets againstcoalescence and thereby providing physical stability to the emulsionduring processing and storage (McClements, 2005). A wide range ofemulsiers is considered as food grade such as small molecularweight surfactants, phospholipids and biopolymers (Krog & Sparso,2004). Nevertheless, food industry has presented a growing interestin the replacement of synthetic emulsiers by natural ones, such aspolysaccharides and proteins (Garti, 1999). The study of emulsifyingproperties of proteins, as casein, whey protein, soybean protein and

* Corresponding author. Tel.: 55 19 35214047; fax: 55 19 35214027.

Contents lists availab

r

ls

Food Hydrocolloids 25 (2011) 604e612E-mail address: rosiane@fea.unicamp.br (R.L. Cunha).water) where one of the liquids is dispersed in the other assmall (0.1e100.0 mm) spherical droplets. Emulsions are thermody-namically unstable systems, but they can be kinetically stabilized(without phase separation) for a reasonable period of time by addingsubstances known as emulsiers and/or thickening agents prior tohomogenization (McClements, 2005).

Emulsifying processes, such as rotorestator, high pressure,membrane or ultrasonic homogenization, can also play an impor-tant role in emulsion stability by reducing the droplet size. Inrotorestator devices, oil and water are mixed at very high rotation

narrow droplet distribution. In this procedure the pressurized liquidis forced through a valve that causes a sudden sharp constrictionin the ow. The pressure energy applied at the valve is transferredinto kinetic energy and the particle break-up is initiated bya combination of turbulence and laminar shear (Dickinson, 1992).The homogenization pressure is usually between 5 and 50 MPa, butit may reach 700 MPa (Floury, Belletre, Legrand, & Desrumaux,2004; Jafari et al., 2008; Marie, Perrier-Cornet, & Gervais, 2002).

During emulsion formation, stabilizers with surface activityare rapidly adsorbed onto the newly formed oilewater interface,1. Introduction

Emulsions consist of two immis0268-005X/$ e see front matter 2010 Elsevier Ltd.doi:10.1016/j.foodhyd.2010.07.018trostatically stable emulsions at pH 3.5. In contrast, emulsions at higher pH values (4.5, 5.5 and 7.5) showeda microscopic three-dimensional network responsible for their stability at protein contents higher than1.0% (w/w). The emulsions at pH 3.5 homogenized by high pressure (up to 100 MPa) showed a decrease insurface mean diameter (d32) with increasing pressure and the number of passes through the homogenizer.These emulsions showed droplets with lower dispersion and d32 between 1.00 and 4.05 mm, six timeslower than values observed for primary emulsions. The emulsions presented shear-thinning behavior andlower consistency index and viscosity at higher homogenization pressures. In addition, the emulsionsshowed a less structured gel-like behavior with increase in homogenization pressure and number ofpasses, since the pressure disrupted the collagen ber structure and the oil droplets. The results of thiswork showed that the collagen ber has a good potential for use as an emulsier in the food industry,mainly in acid products.

2010 Elsevier Ltd. All rights reserved.

iquids (usually oil and

produced with this system (Jafari, Assadpoor, He, & Bhandari, 2008).On the other hand, high-pressure homogenizers are used to producestable emulsions with droplet diameter smaller than 1 mm andKeywords:

The phase separation and droplet size of the emulsions prepared using the rotorestator device (primaryemulsion) decreased with protein concentration and reduction in pH, allowing the production of elec-Accepted 21 July 2010 pressure homogenizer). The stability, microstructure and rheology of the O/W emulsions were measured.Emulsifying properties of collagen berconcentration and homogenization pres

R.C. Santana, F.A. Perrechil, A.C.K. Sato, R.L. Cunha*

Department of Food Engineering, Faculty of Food Engineering, University of Campinas (

a r t i c l e i n f o

Article history:Received 17 March 2010

a b s t r a c t

The emulsifying propertiesunder different conditions

Food Hyd

journal homepage: www.eAll rights reserved.Effect of pH, proteinre

CAMP), P.O. Box 6121, 13083-862 Campinas, SP, Brazil

collagen bers were evaluated in oil-in-water (O/W) emulsions producedH, protein content and type of emulsication device (rotorestator and high-

le at ScienceDirect

ocolloids

evier .com/locate/ foodhyd

drocgelatin, has been the subject of several recently published researchpapers (Keerati-u-rai & Corredig, 2009; Lizarraga, Pan, Aon, &Santiago, 2007; Neirynck, Van lent, Dewettinck, & Van der Meeren,2007; Surh, Deckes, & McClements, 2006).

Collagen is a proteinwidely used in food industries to improve theelasticity, consistency and stability of foods, although its use has onlybeen carried out in an empirical way (Olivo & Shimokomaki, 2002).Collagen ber is a new ingredient obtained from collagen in its crudeform, while gelatin is produced from collagen hydrolysis (Nicoleti &Telis, 2009; Wolf, Sobral, & Telis, 2009). Collagen ber is obtained byscraping pieces of dermis and subcutaneous tissue. This materialis previously submitted to chemical treatment for defatting, dried ata mild temperature and ground for the collagen ber production.During the last step, two fractions are generated according to theparticle size: the ner one is classied as collagen powder, while thecoarser one corresponds to collagen bers. Differences in particleshape between the fractions can also be expected (Nicoleti & Telis,2009). More recent studies have shown the gelation properties ofcollagen ber (Mximo & Cunha, 2008), and its use as a rawmaterialfor the production of self-biocomposite lms (Wolf et al., 2009).However, the emulsifying properties of collagen ber have not yetbeen extensively explored, in spite of their traditional use as a waterand fat binder in meat products (Asghar & Henrickson, 1982).

In this context, the purpose of this study was to investigate thepotential use of collagen ber as an emulsier in food products,evaluating the effects of pH, protein content, emulsifying device(rotorestator and high-pressure homogenizer), homogenizationpressure (20e100 MPa) and the number of homogenization cycleson the phase separation, droplet size and rheology of the emulsionsstabilized by collagen ber.

2. Material and methods

2.1. Material

Collagen ber extracted from bovine hides was kindly providedby NovaProm Food Ingredients Ltda (Guaiara, SP, Brazil). Thedetailed physico-chemical characterization of collagen ber,including its amino acid composition, was described by Nicoletiand Telis (2009), who found a protein content close to 86% (w/w).Soybean oil (Bunge, Brazil) was purchased from a local market andall chemicals used were of analytical grade (Sigma ChemicalCompany, St. Louis, USA).

2.2. Emulsion preparation

Initially, collagen ber dispersions (aqueous phase) wereprepared using deionized water with gentle magnetic stirring atroom temperature for 1 h. The pH of the samples was constantlyadjusted to the desired value with 1.0 M HCl or 1.0 M NaOH solu-tions. Sodium azide (0.1%w/w) was added to prevent microbialgrowth.

Oil-in-water emulsions (O/W) were prepared using twodifferent emulsifying processes: (i) primary emulsions wereprepared by mixing the collagen ber dispersions with 10% (w/w)soybean oil using a rotorestator device (Ultra-Turrax mixer, modelT18 basic, Germany) at 20,000 rpm for 4 min. These emulsionsshowed a nal protein content of 0.5, 1.0, 2.0 and 3.0% (w/w) atdifferent pH values (3.5, 4.5, 5.5 and 7.5); (ii) the primary emulsionsprepared in the rotorestator device and composed by 0.5% protein(w/w) at pH 3.5, were processed in a two-stage high-pressurehomogenizer (NS1001L2K-PANDA 2K, Niro Soavi S.p.A., Parma,Italy). The effect of homogenization pressure on emulsion proper-ties was investigated by applying different pressures in the rst

R.C. Santana et al. / Food Hystage of the homogenizer (20e100 MPa). The pressure in thesecond stage was xed at 5 MPa, since a low pressure homoge-nizing valve is conventionally used to increase cavitation anddecrease droplet recoalescence phenomenon (Freudig, Tesch, &Schubert, 2003; Mohan & Narsimhan, 1997). The emulsions wererun through the high-pressure homogenizer once or twice (recy-cling), and the temperature was measured immediately after.

All systems were evaluated according to its stability and particlesize. Rheological analysis was also done for emulsions homoge-nized at high pressure.

2.3. Emulsion properties

2.3.1. StabilityImmediately after the emulsifying process, aliquots of the emul-

sions prepared using the rotorestator device and the high-pressurehomogenizer were placed in 10 mL and 50mL graduated cylinders,respectively. The stability of the emulsions to occulation/coales-cence was evaluated by visually monitoring the development ofa bottomphase during 7 days of storage at room temperature (25 C).

The creaming index (CI %) of the emulsions was expressed as CI(%) (Vs/Vi) 100, where Vi represents the initial volume of theemulsion and Vs the volume of clear serum formed at the bottom ofthe tubes (Keowmaneechai & McClements, 2002).

The inuence of the pH value and protein content on thecreaming process was analyzed using a rst order kinetics equationgiven as CI (%) CIeq (1 expkt), where CIeq is the equilibriumcreaming index, t is the time and k is the creaming rate in h1.

2.3.2. Optical characterization and size distribution of oil dropletsThe microstructure of the emulsions was studied using a Carl

Zeiss light microscope Model mf-AKS 24 36 EXPOMET (Zeiss,Germany). Emulsions homogenized using the rotorestator deviceand high-pressure equipment were analyzed immediately after theemulsifying process and after 7 days of storage, respectively. Thesamples were poured onto microscope slides, covered with glasscover slips and observed at a magnication of 40 and 100.At least 20 imageswere taken for each sample and the best 10 sharpimages were analyzed using the public domain software ImageJ v1.36b (http://rsb.info.nih.gov/ij/).

Micrographs of the emulsion prepared using the rotorestatordevice were analyzed according to Perrechil and Cunha (2010). Thepictures were transformed into 8-bit grey scale binary images with640e480 pixels and then segmented by thresholding. The greylevel used to threshold the image was the median of the grey levelhistogram of each image. During this process, the pixels were onlydeemed as detected if their grey valuewas lower than the thresholdsetting (Pugnaloni, Matia-Merino, & Dickinson, 2005). Dropletsconnected to image border were suppressed of image analysis.

Micrographsof theemulsionshomogenizedbyhighpressure couldnot be analyzed according to Perrechil and Cunha (2010) and Pugna-loni et al. (2005), since theirdroplet diameterswere less than5 mm, toosmall to allow reliable droplet identication. Thus these images wereanalyzed by measuring at least 500 droplets (one by one).

After conversion of the pixel-scale into microns using a scalingfactor, surface mean diameter (d32) of the emulsions was calculatedas d32

P(nidi3)/

P(nidi2), where ni was the number of particles

with diameter di.The droplet size distributions were statistically compared

with a probability density function of a log-normal distribution(Equation (1)) using the non-parametric KolmogoroveSmirnov testand the Statistica 5.5 software (Statsoft Inc., Tulsa, USA).

f di 1 p exp

"ln di ln dg2

2

#(1)

olloids 25 (2011) 604e612 605ln sg 2p 2 ln sg

where dg and sg were the geometric mean and standard deviationof the geometric mean, respectively, as given by Equations (2) and(3) (McClements, 2005):

ln dg X

ni ln di=N (2)

ln sg Xh

niln di ln dg

2i=N

r(3)

where N is the total number of droplets.

2.3.3. Rheological measurementsRheological measurements were carried out using a Physica

MCR301 rheometer (Anton Paar, Germany). Samples obtainedimmediately after the homogenization process were placed ina parallel plate geometry (with a 0.6 mmgap)made of glass (50 mmdiameter) or stainless steel (75 mm diameter). Steady shear andoscillatory measurements were carried out in triplicate at 25 C.The rheological analyses were not applied to the primary emulsionshomogenized in the rotorestator device, since most of themshowed phase separation only a few minutes after preparation.

Flow curves were obtained using an upedowneup step programwith a shear rate range between 0 and 300 s1. The rheologicalbehavior of the emulsions was analyzed according to the Power-Lawmodelandexpressedass k _gn,wheres is theshearstress (Pa), _gthe shear rate (s1),k the consistency index (Pa sn) andn theowindex.

Oscillatory measurements were used to determine the complexshear (G*), storage(G0)andloss (G00)modulioveranascendingfrequency

Tukey Procedure and the statistical analyses were applied using thesoftware Statistica 5.5 (Statsoft Inc., Tulsa, USA).

3. Results and discussion

3.1. Emulsions homogenized using the rotorestator device(primary emulsions)

The effects of pH (3.5, 4.5, 5.5 and 7.5) and protein content(0.5, 1.0, 2.0 and 3.0%w/w) were evaluated through their effects oncreaming kinetics and droplet size of the primary emulsionsprepared using the rotorestator device.

3.1.1. Creaming kineticsFig. 1 shows the creaming kinetics of the emulsions with storage

time, veried by visual observations. As can be seen from theproles, phase separation usually occurred after short times (in therst 30 min) for very high creaming rates.

In general, emulsion stability improved with increase in proteinconcentration and reduction in pH, reducing the equilibriumcreaming index (CIeq) and creaming rate (k) (Table 1). The moststable emulsions were observed at pH 3.5, making it possible toproduce acid emulsions stabilized by a protein. Emulsions at pH 3.5with protein concentrations higher than 0.5% (w/w) showed nosigns of serum separation even after 7 days of storage. The openingof collagen triple helices at pH 3.5 modied the protein surfacehydrophobicity, and could be partially responsible for the better

00 20 40 60

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612606rampof0.1e10 Hz.Allmeasurementswerecarriedoutwithin the linearviscoelastic region (deformation less or equal than 1.0%).

2.4. Statistical analysis

The measurements were done in triplicate. Signicant differ-ences (p< 0.05) between treatments were determined by the

0

20

40

60

80

0 20 40 60

Time (min)

CI (%

v/v)

A

0

20

40

60

80

0 20 40 60

Time (min)

CI (%

v/v)

CFig. 1. Effect of pH and protein content on the phase separation of emulsions stabilized by1.0% ( ), 2.0% (e6e) and 3.0% ( ). pH: 3.5 (A), 4.5 (B), 5.5 (C) and 7.5 (D).Time (min)emulsifying properties of the collagen ber at this low pH.However, electrostatic interactions between the emulsion dropletsare probably the most important inuence of the pH. In emulsionsstabilized by proteins, the droplet surface is charged if the pH isfar from the protein pI. As the collagen ber pI is between 6.5 and8.5 (Neklyudov, 2003), the droplets are positively charged at pH3.5, which led to a strong electrostatic repulsion between them,

0

20

40

60

80

0 20 40 60

Time (min)

CI (%

v

/v)

B

20

40

60

80

CI (%

v

/v)

Dcollagen bers in the rst 60 min after processing. Protein concentration: 0.5% (eBe),

preventing droplets from coming close enough together to aggre-gate and coalesce (McClements, 2005).

On the other hand, emulsions maintained at pH values close tothe protein pI show uncharged surface droplets and the repulsiveforcesmay no longer be strong enough to prevent the droplets fromaggregating (McClements, 2005), so that these droplets shouldbe attracted due to hydrophobic and Van der Walls forces (Chen,Dickinson, & Edwards, 1999). At this pH condition, the higherstability observed with increased protein content can be explainedby the viscosity enhancement and steric stabilization of thecollagen ber, decreasing droplets mobility and consequent coa-lescence and destabilization. In spite of the high pH, emulsions at

pH 7.5 and stabilized by more than 0.5% (w/w) protein, showed anintermediary creaming rate (k) that could be explained by thepartial solubility of the collagen ber at its pI (Mximo & Cunha,2008), retarding droplet mobility due to steric hindrance.

3.1.2. Microstructure and droplet sizeThe microstructures of the emulsions prepared using the

rotorestator device and stabilized by collagen bers are shownin Fig. 2, revealing polydisperse emulsions with a wide range of oildroplet sizes. Droplet size distribution is an important parameter forsome emulsion properties such as shelf life and texture, and thus itscontrol and measurement is important (McClements, 2005).

Table 1Equilibrium creaming index (CIeq % v/v) obtained after 7 days of emulsion storage and creaming kinetics as described by the parameter of the rst order kinetics equation (k1)tted to the creaming index (R2> 0.90) of the emulsion homogenized by the rotorestator device during seven days of observation.

% Protein (w/w) Equilibrium creaming index (CIeq (% v/v)) Creaming kinetics (k (h1))

pH pH

3.5 4.5 5.5 7.5 3.5 4.5 5.5 7.5

0.5 1.00Aa 63.00Ab 56.33Ac 65.67Ab 0.02Aa 44.70Ab 34.58Ac 46.42Ab

1.0 0.00Aa 54.33Bb 53.67Ab 57.50Bb 0.00Aa 19.54Bbc 25.69Bb 11.63Bd

2.0 0.00Aa 15.33Cb 29.00Bc 29.00Cc 0.00Aa 2.58Cb 6.53Cc 1.77Cb

3.0 0.00Aa 0.00Da 11.67Cb 13.33Db 0.00Aa 0.00Da 1.95Db 0.69Cab

Different superscript letters indicate signicant difference (p> 0.05). Capital letters compare differences within a column and small letters compare differences betweendifferent pH values for the same creaming parameter.

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612 607Fig. 2. Micrographs of O/W emulsions produced using the rotorestator device and stabilized by collagen bers. Scale bar 50 mm.

However, a considerable amount of the process energy added in thisemulsication method is dissipated in the form of heat, so that theproduction of monodispersed emulsions with a small droplet size isdifcult (Anton, Benoit, & Saulnier, 2008).

The emulsions showed structural differences depending on thepH value. True emulsions were observed at pH 3.5 (pH far from thatof the proteinpI),with droplets surrounded by a continuous aqueousphase. On the other hand, emulsions produced at higher pH valuesshowed a macroscopically three-dimensional network formed dueto the absence of electrostatic repulsion between droplets. In addi-

pressure is shown in Fig. 4, revealing that the droplet size and itspolydispersity decreased with increase in pressure and number ofpasses through the homogenizer. This occurred because the higherhomogenization pressure led to an increase in impact forces thatact on the droplets, causing disruption of the interfacial membranes(McClements, 2005) with a consequent increase in the interfacialarea and interaction between oil and emulsier (Floury, Desru-maux, & Lardires, 2000). The effect of the number of passes can beassociated with the increase in residence time of the emulsier inthe homogenizer valve, allowing for an improved adsorption ontothe droplet surface before their collision and coalescence (Flouryet al., 2000).

The surface mean diameter (d32) varied between 1.00 and4.05 mm (Fig. 5), showing a considerable reduction in droplet sizeup to 60 MPa, followed by a slight additional decrease up to100 MPa/1 pass. No further droplet reduction was observed withtreatments above 60 MPa/2 passes, and in such a way that thispoint, where the surface mean diameter reached a plateau ataround 1.00 mm, can be identied as the critical limit. The surface

R.C. Santana et al. / Food Hydro608tion, a high content of insoluble protein can be clearly observed inthe emulsions at pH 7.5, 5.5 and 4.5. Thus, collagen ber probablyacts as a stabilizer at these conditions. It was evident that emulsionsformed at higher pH values containing more than 1.0% (w/w) ofprotein, were stabilized by a three-dimensional network composedof an almost intact collagenber structure that reduced the dropletsmovements and decreased the CIeq.

Table 2 shows the average droplet size (d32) of the emulsionsprepared using the rotorestator device. It was only possible toevaluate the emulsions produced at pH 3.5 and under someconditions at pH 4.5, due to difculties in visualizing the droplets inthe others conditions, as mentioned before. Surface mean diameterof the oil droplets varied between 6.71 and 12.76 mm. In agreementwith the stability tests, the smallest droplet sizes were observed atpH 3.5 with protein content above 0.5% (w/w), conrming emul-sifying properties of collagen ber at low pHs.

3.2. Emulsions homogenized by high pressure

The effects of homogenization pressure (20e100 MPa) and thenumber of passes through the homogenizer were evaluated usingO/W emulsions composed of 10.0% (w/w) soybean oil and 0.5%(w/w) collagen ber protein at pH 3.5, which was the best pHcondition to obtain stable emulsions using the rotorestator device.

3.2.1. Temperature rise during high-pressure homogenizationThe temperature of the emulsions increased linearly with the

pressure and number of passes through the homogenizer (Fig. 3),in agreement with Desrumaux and Marcand (2002) and Floury,Desrumaux, Axelos, and Legrand (2003). The rise in temperaturedepends on the uid composition (Corts-Muoz, Chevalier-Lucia,& Dumay, 2009) and on the conguration and heat capacity of thehomogenizer itself (Sandra & Dalgleish, 2005). The distinctwarming up of the uid was due to viscous stress caused by thehigh velocity of the uid ow, which then impinged on the valve(McClements, 2005).

Heating during homogenization occurs during a short period oftime, and its contribution to modication of the macromoleculesanddroplet size is uncertain (Floury et al., 2003). Heat can contributeto the production of small droplets by decreasing the interfacialtension between the oil and water, or by reducing the viscosityof both phases. However, proteins used as emulsiers can losetheir functional properties when heated above the denaturation

Table 2Surface mean diameters d32 (mm) for emulsions emulsied using the rotorestatordevice and stabilized by collagen bers.

Protein concentration(%w/w)

pH

3.5 4.5 5.5 7.5

0.5 12.76 2.26x e e e1 8.60 1.65y e e e2 6.71 1.19y 9.88 1.36xy e e3 7.76 1.37y 11.71 1.56x e eDifferent superscript letters indicate signicant difference (p> 0.05).temperature. In these conditions, collagen is disintegrated froma triple helical structure to random coils, accompanied by changes inits physical properties (Usha & Ramasami, 2004). At pH 3.0, thecollagen starts to denature at 39.5 C (Tonset), with denaturationtemperature (maximum peak) of 52.5 C (Tpeak) (Wolf et al., 2009).

Thus some structural modications of the collagen bers couldhave happened in the emulsions homogenized by pressures above60 MPawith 1 pass, or 40 MPawith 2 passes, although no emulsionreached the peak temperature for collagen denaturation (Fig. 3).

3.2.2. Creaming indexAll the emulsions homogenized by high pressure showed

good stability, with no signs of phase separation in 10 mL cylinders(diameter 15.5 mm, height 65 mm). In order to reduce walleffects, stability tests of these emulsions were also carried out in50 mL cylinders (internal diameter 25 mm, height 95 mm),since it is well known that the conning wall can exert anextra retardation effect on a spherical particle settling in a liquid(Chhabra, Agarwal, & Chaudhary, 2003). Under these conditions,the emulsion processed under the mildest conditions (20 MPawithone pass), which showed a non homogeneous aspect, presented10% (v/v) of serum phase separation after 7 days of storage. All theother emulsions showed a homogeneous aspect and no phaseseparation even in 50 mL cylinders.

3.2.3. Microstructure and droplet size distributionThe microstructure of the emulsions homogenized by high

30

35

40

45

50

55

0 20 40 60 80 100 120

Pressure (MPa)

Tem

pera

ture

(C) Range of

collagen denaturation

Tpeak

Tonset

Fig. 3. Temperature of emulsions as a function of homogenization pressure. Number ofpasses through the homogenizer: (C) 1 and (-) 2. Tonset (initial denaturation tem-perature) 39.5 C and Tpeak (denaturation temperature) 52.5 C (Wolf et al., 2009).

colloids 25 (2011) 604e612mean diameter can be related to the homogenization pressure (P)

exponent m obtained was around 1. The slight decrease in theabsolute value for m (turbulence increase) when the emulsionswere passed twice through the homogenizer, can be explained fromthe reduction in oil droplet packing (Fig. 4), with a consequentdecrease in emulsion viscosity (Fig. 7 and Table 4).

The droplet size distribution of the emulsions was welldescribed by a log-normal distribution (Fig. 6), as reported by otherauthors (Ambrosone & Ceglie, 1997; Coupland & McClements,2001; McClements, 2005; McClements & Coupland, 1996). Table 3

sure and stabilized by 0.5% (w/w) of protein. Scale bar 10 mm.

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612 609using the Power-Law adjustment (d32f Pm). The exponent m ofthe Power Law has been empirically associated with Reynoldsnumber (Re), which depends on the valve dimension, liquidviscosity and homogenization pressure. This parameter showsvalues between 0.6 and 1.0, depending on the ow regime (Flouryet al., 2003). For a large homogenizer and a low uid viscosity, theow regime is predominantly turbulent-inertial and df P0.6.For a large homogenizer and a high uid viscosity, the ow regimeis predominantly turbulent-viscous and d32f P0.75. For smallhomogenizers such as those used on a bench-scale, the ow regimemay even be laminar-viscous and d32f P1.0 (McClements, 2005).

Several authors have discussed the relationship between pres-sure and droplet size. Walstra and Smulders (1997) obtainedd32f P

0.9 for homogenization at P 15 MPa, while Floury et al.(2003) obtained d32f P0.48 for homogenization at P 350 MPa.For the emulsions stabilized by collagen bers and emulsied atP 100 MPa, values of d32f P1.08 (R2 0.95) and d32f P0.90(R2 0.90) were found for the samples homogenized using 1 and 2passes, respectively. It should be noticed that the surface meandiameter of the emulsions homogenized at 20 MPawith 1 pass was

Fig. 4. Micrographs of emulsions homogenized by high presnot included in the Power-Law tting, since this emulsion showedphase separation. From the obtained results it can be estimatedthat the ow in the homogenizer was laminar-viscous, since the

0

1

2

3

4

5

0 20 40 60 80 100 120Pressure (MPa)

d 32

( m

)

Fig. 5. Effect of homogenization pressure and number of passes on the surface meandiameters (d32) of the emulsions homogenized at high pressures. Number of passes:(C) 1 and (-) 2.A

10

15

20

25

Freq

uenc

y (%

)0

5

0.010.11.0

Diameter (m)

B

0

5

10

15

20

25

0.010.11.0Diameter (m)

Freq

uenc

y (%

)

Fig. 6. Effect of homogenization pressure and number of passes on the droplet sizedistribution in the emulsions homogenized using high pressures. Pressure (MPa): ( )20, ( ) 40, ( ) 60, (e e e) 80 and ( ) 100. Number of passes: (A) 1 and (B) 2.

a submicron scale, reaching values below 1.0 mm. Emulsionshomogenized at 60 MPa showed a high droplet size frequencyat the lowest values observed (between 0.2 and 0.4 mm), whilethe emulsions homogenized at 80 and 100 MPa showed lowerpolydispersity.

3.2.4. RheologyThe ow curves of all emulsions homogenized at high pressure

and stabilized using collagen bers showed shear-thinning

Table 3Parameters obtained from the log-normal distribution of the droplet size.

Homogenizationpressure (MPa)

Geometric meandg=mm

Standard deviation(sg/mm)

1 Pass 2 Passes 1 Pass 2 Passes

20 2.83 2.66 1.65 1.4940 2.56 1.90 1.71 1.5760 1.36 0.70 1.75 1.5480 1.45 0.85 1.57 1.25100 0.93 0.87 1.46 1.28

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612610A

8

10

(Pa)shows the geometric mean and its standard deviation of thegeometric mean as obtained from the log-normal distribution. Inagreement with observations of the micrographs, the dropletsize dispersion (described by a log-normal standard deviation)decreased with increase in homogenization pressure and numberof passes, specially the emulsions homogenized with pressures of80 and 100 MPa, which showed a high frequency of droplets on

0

2

4

6

0 50 100 150 200 250 300

Shear rate (s-1)

Shea

r st

ress

B

0

2

4

6

8

10

0 50 100 150 200 250 300

Shear rate (s-1)

Shea

r st

ress

(Pa)

Fig. 7. Shear stress versus shear rate for the emulsions homogenized using highpressures and stabilized by collagen bers. Homogenization pressure (MPa): 20 (,),40 (e), 60 (6), 80 (B) and 100 (>). Number of passes: (A) 1 and (B) 2.

Table 4Rheological parameters obtained from the Power-Law model and apparent viscosity at 10

Rheological parameter Homogenization pressure (MPa) with 1 pass

20 40 60 80

n (e) 0.53Aa 0.48Ba 0.52Aa 0.53Aa

k (Pa sn) 0.25Aa 0.56Ba 0.48Ca 0.38Da

h100 (mPa s) 27.6Aa 51.1Ba 51.3Ba 43.4Ca

Different superscript letters indicate signicant difference (p> 0.05), capital letters comsmall letters compare differences between number of passes at the same homogenizatiobehavior (Fig. 7). The decrease in apparent viscosity of the emul-sions with increasing shear rate could be attributed to the defor-mation and disruption of clusters or aggregates of droplets, andtheir ordering within the ow eld (McClements, 2005).The Power-Law was tted to the data (Table 4), showing a gooddetermination coefcient (R2> 0.999) for all emulsions. Table 4shows the apparent viscosity at 100 s1 of the emulsions homog-enized by high pressure and stabilized by collagen bers.

The emulsion homogenized at 20 MPa with 1 pass showed lowvalues for the consistency index (k) and apparent viscosity.The rheological measurements were probably not reliable, since itshowed a weak structure and phase separation. In general, theemulsions homogenized at high pressure showed higher values forthe ow index and lower values for the consistency index withincrease in pressure and number of passes. Moreover, by comparingthe up and down ow curves for emulsions homogenized at higherpressures (80 and 100 MPa) (results not shown), less hysteresis wasobserved, suggesting that the emulsion structure was broken in thehomogenization process.

The apparent viscosity increased slightly in the emulsionshomogenized at pressures from 20 MPa/2 passes to 60 MPa/2passes. The increase in viscosity with reduced droplet size could beattributed to an increase in hydrodynamic interactions between thedroplets, since the mean separation distance between the dropletsdecreases when the droplet size is reduced (Pal, 2000). Moreover,a greater amount of absorbed protein or more tightly packedproteins at the oil-in-water interface can increase the emulsionviscosity (Innocente, Biasutti, Venir, Spaziani, & Marchesini, 2009).

On the contrary, emulsions homogenized at 80 and 100 MPashowed a reduction in the viscosity with decreasing droplet size, asobserved by Desrumaux and Marcand (2002). In this case, smallerdroplet sizes led to lower internal resistance than larger onesor linear chains of droplets (Schimidt & Smith, 1989). Thus, thebehavior of the viscosity may be well related to mean and dropletsize distribution. As can be observed in Figs. 5 and 6 and Table 4, thesurface mean diameter decreased with decrease in pressure up to60 MPa, while droplet size polydispersity decreased considerably at80 and 100 MPa. Floury et al. (2003) and Schulz and Daniel (2000)showed that the decrease in emulsion viscosity was related to thereduction in food protein functionality caused by the homogeni-zation pressure, although this was not clear veried in the presentwork.

Emulsions homogenized at high pressures and stabilizedusing collagen bers showed a gel-like behavior, with the storagemodulus (G0) greater than the loss modulus (G00) in all cases (data

0 s1 for emulsions homogenized at high pressure and stabilized by collagen bers.

Homogenization pressure (MPa) with 2 passes

100 20 40 60 80 100

0.52Aa 0.46Ab 0.50Bb 0.50Bb 0.67Cb 0.69Cb

0.40CDa 0.53Ab 0.50Aa 0.54Aa 0.09Bb 0.05Bb

43.2Ca 44.3Ab 48.5ABa 53.3Ba 19.1Cb 12.3Dbpare differences between homogenization pressure for the same number of passes;n pressure.

a)droc1

G*

(PA

0,1

1

10

100

0111.0Frequency ( Hz)

G*

(Pa)

B

10

100

R.C. Santana et al. / Food Hynot shown). An increase in homogenization pressure and numberof passes decreased the complex modulus (G*) and increased thedependence of G0 and G00 on the frequency (Fig. 8), indicating thatthis process disrupted the collagen ber structure as well as that ofthe oil droplets, resulting in less structured emulsions.

4. Conclusions

The study of the primary emulsions showed that electrostaticinteractions were responsible for the emulsion stability at pH 3.5.This occurred because the pI of the collagen bers is between 6.5and 8.5, which is considerably higher than those of conventionalprotein emulsiers such as soybean, casein andwhey proteins. Withrespect to the primary emulsions at higher pH values (4.5, 5.5 and7.5), the increase in sample stability was only observed as a functionof the protein content. At higher pH values, the collagen bersshowed steric hindrance, forming a lm around the droplets andreducing their mobility and consequent coalescence. The use ofhigh-pressure homogenization produced stable and less structuredemulsions, since this process disrupted the collagen ber structureand the oil droplets. Up to a pressure of 60 MPa, the reduction insurface mean diameter led to an increase in emulsion viscositycaused by an increase in hydrodynamic interactions between thedroplets. On the other hand, at higher pressures (80 and 100 MPa),a considerable decrease in droplet size polydispersity was apparent,with a consequent reduction in emulsion viscosity. Finally, theresults of this work allowed for the elucidation of some of theinteraction mechanisms of collagen bers, and their inuence on

0,10111.0

Frequency ( Hz)Fig. 8. Inuence of homogenization pressure and number of passes on the complexmodulus (G*) of the emulsions homogenized using high pressure and stabilized bycollagen bers. Pressure: 20 (>), 40 (,), 60 (B), 80 (6) and 100 MPa (e). Number ofpasses: (A) 1 and (B) 2.the stability, structure and rheology of emulsions, showing that thebest conditions to use this ingredient would be at acid pH values.

Acknowledgments

The authors would like to thank CNPq for their nancial support.

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R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612612

Emulsifying properties of collagen fibers: Effect of pH, protein concentration and homogenization pressureIntroductionMaterial and methodsMaterialEmulsion preparationEmulsion propertiesStabilityOptical characterization and size distribution of oil dropletsRheological measurements

Statistical analysis

Results and discussionEmulsions homogenized using the rotorstator device (primary emulsions)Creaming kineticsMicrostructure and droplet size

Emulsions homogenized by high pressureTemperature rise during high-pressure homogenizationCreaming indexMicrostructure and droplet size distributionRheology

ConclusionsAcknowledgmentsReferences