International Dairy Journal 14 (2004) 777–782

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doi:10.1016/j.ida

Flow and creep compliance properties of reduced-fat yoghurtscontaining protein-based fat replacers

C. Lobato-Callerosa, O. Mart!ınez-Torrijosa, O. Sandoval-Castillaa,J.P. P!erez-Orozcob,c, E.J. Vernon-Carterb,*

aDepartamento de Preparatoria Agr!ıcola, Universidad Aut !onoma Chapingo, Km 38.5 Carretera M!exico-Texcoco, Texcoco 56230, MexicobArea de Ingenier!ıa Qu!ımica, Universidad Aut !onoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina, 09340, D.F. Mexico

cDepartamento de Ingenier!ıa Qu!ımica y Bioqu!ımica, Instituto Tecnol !ogico de Zacatepec, CP. 62780, Zacatepec, Mor., Mexico

Received 12 September 2003; accepted 27 February 2004

Abstract

The flow and creep compliance properties of reduced-fat yoghurts containing whey protein concentrate (WPC), microparticulated

whey protein, or a blend of both fat replacers were determined and compared to those exhibited by a full-fat yoghurt (FFY). The

flow behaviour of all the yoghurts was described by the Ellis equation. Rheological parameters such as instantaneous compliance

(J0), mean compliance (Jm), mean retardation time (tm), and Newtonian viscosity (ZN) were useful to explain structural

characteristics and changes in the protein network of the reduced-fat yoghurts. The yoghurt made with WPC showed flow and

viscoelastic properties that resembled more closely those of the FFY.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Protein based fat replacers; Reduced-fat yoghurts; Flow properties; Creep compliance properties

1. Introduction

Health concerns have led consumers worldwide toreduce consumption of foods perceived as high in fat,which has opened way to a growing market of foodsconsidered as healthy, with good mouthfeel andincorporating natural products only. Thus, yoghurtproducers are motivated to market low-fat productswith natural ingredients. Changes in the fat content ofyoghurts modify their rheological behaviour. Wilkinson,Guinee, and Fenelon (1999) developed a laboratoryfermented milk model system and studied the effects ofmilk components on the rheology of yoghurt. Increasingthe fat content within the range of 0.37–4%, whilemaintaining the protein constant, resulted in increases inthe storage modulus and apparent viscosity. Keogh andO’Kennedy (1998) reported that yoghurts obtained byvarying the level of protein, fat and hydrocolloids,provided a large range of consistencies and brittlenesslevels. Protein was the most effective component at

g author. Tel.: +52-55-8044648; fax: +52-55-

s: jvc@xanum.uam.mx (E.J. Vernon-Carter).

front matter r 2004 Elsevier Ltd. All rights reserved.

iryj.2004.02.012

increasing consistency and fat was next in effectiveness.Sandoval-Castilla, Lobato-Calleros, Aguirre-Manduja-no, and Vernon-Carter (2004) reported that reduced-fatyoghurt exhibited lower tension and firmness than full-fat yoghurt, as a result of lower number of fat globulesacting as structure promoters of the protein network.Heating of skim milk to 75–90�C before inoculation isessential for the proper development of yoghurtstructure. It results in firmer yoghurts than those madefrom unheated milk (Aguilera & Stanley, 1999). Heattreatment of milk at X80�C greatly increased thestorage modulus (G0) of acid milk gels compared withgels made from unheated milk (Lucey, Munro, & Singh,1998), and there were no major differences in themicrostructure of acid milk gels formed in the range 80–90�C (Lucey, Teo, Munro, & Singh, 1998). Homo-genised fat globules directly participate in the acidcoagulation process and finally become an integral partof the network structure (Buchheim & Dejmek, 1997).

When formulating reduced-fat yoghurt one must seekthe reinforcement of the protein network to build up thestructure. A potential good solution is to use pure milkingredients such as whey proteins. Mistry and Hassan(1992) reported that good quality non-fat yoghurts

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could be produced by supplementing skim milk withhigh milk protein powder up to 5.6%. Modler andKalab (1983) found that yoghurts prepared with wheyprotein concentrates (WPC) were generally softer thanyoghurt prepared from casein-based ingredients.Sandoval-Castilla et al. (2004) reported that reduced-fat yoghurts showing similar instrumental texturecharacteristics to full-fat yoghurt (FFY) could beobtained when supplementing low-fat milk with WPC.When the low-fat milk was added with microparticu-lated whey protein (MWP) the tension and firmnesstextural characteristics of the yoghurt were slightlylower than those of the full-fat yoghurt.

The ‘‘gel-like’’ effects that proteins and stabilisersimpart to liquids, which can be used by food scientists toimprove texture and stability, can be best investigatedby employing proper rheological measurements (Schenz,1997). Measurement of the flow curves of these fluids ona log viscosity versus log shear stress (shear rate) plot,over a wide range of shear stress (shear rate), allows thedetermination of the ‘‘structure point’’, i.e. the stress(shear rate) below which structure builds. Viscoelasticproperties of yoghurt can be measured by creepcompliance test, in which the sample deformation dueto an imposed constant stress is recorded as a functionof time. Afterwards, the deformation is converted tocreep compliance (deformation/unit of constant stress)and the rheological parameters: compliances, retarda-tion times and viscosities are calculated from creepcurves (Rao, Kash, Cooley, & Barnard, 1987). The creepexperiment results, when taken together with the flowcurve results, provide the basis for understanding thenature of the weak gel structure-building process thatoccurs due to chain entanglement or associationprocesses in fluid foods (Schenz, 1997). Furthermore,creep compliance-time studies carried out within thelinear-viscoelastic region, have the advantage of avoid-ing destruction in the sample. This permits thedetermination of rheological parameters under condi-tions which approach its conditions at undisturbed stateand, as a consequence, allows a relationship between theresults obtained and the actual structure of the materialto be drawn (Munoz & Sherman, 1990).

The objective of this work was to evaluate the effect ofMWP and WPC upon the flow and the creepcompliance properties of reduced-fat yoghurts, incomparison to FFY.

2. Materials and methods

2.1. Milk and fat replacers

Homogenised and spray-dried whole milk (Nidos)was obtained from Nestle S.A. de C.V. (Mexico), andskim milk powder (Lactomixs) was purchased from

Dilac, S.A. de C.V. (Mexico). The commercial fatreplacers used were: Dairy-los (Cultor Food Science,Mexico), containing 35% of partially denatured WPCand Simplesses 100 (NutraSweet, Mexico), made up of50.5% MWP. Whey protein in Dairy-los is subjected tocontrolled thermal denaturation causing protein unfold-ing and exposure of the hydrophobic regions on thepolypeptide chains, and controlled self aggregation ofmacromolecules through non covalent (i.e. van derWaals) and covalent (i.e. intermolecular disulfideexchange reactions) interactions (Morr & Josephson,1968; Pfizer Food Science Group, 1995). In Simplesses

100 whey protein is heat coagulated forming largeparticles of gel, which are then microparticulated byapplying shear to reduce the coagulating proteins tovery small spheroidal particles (1.0–2.0 mm in diameter)(Lucca & Tepper, 1994). Unlike the WPC’s, the role ofMWP is to simulate fat globules and impart a creamymouthfeel rather than to interact with other milkproteins (Tamime, Kalab, Muir, & Barrantes, 1995).

2.2. Yoghurt premixes

A FFY was made from reconstituted whole milkpowder with 3070.3 g of fat L�1 and 12071 g of totalsolids L�1. Three reduced-fat yoghurt treatments wereelaborated using reconstituted low-fat milk (prepared byblending skim and whole milk powders to obtain1570.1 g of fat L�1 and 12071 g of total solids L�1)added with WPC and MWP on their own and blended.The quantity of fat replacers individually used inmaking yoghurt was based on usage levels recom-mended by the manufacturer: 10 g WPC L�1milk, 10.5 gMWP L�1milk, and for the blend of both fat replacers(5 g WPC+5.3 g MWP)L�1 milk was used. All yoghurtsproduced were set-style.

2.3. Yoghurt preparation

Whole milk and low-fat milk powders were recon-stituted at 30�C with moderate mixing. The dispersionswere refrigerated at 4�C for 24 h, to allow full hydrationof the powders, before usage. Ten litre batches of thereconstituted milk (whole-fat or low-fat) were used forpreparing the yoghurts. Initially, the reconstituted milkswere heated to 30�C, (60 gL�1) sugar was added, and inthe case of the treatments containing fat replacers thesewere added to reconstituted low-fat milk with moderatemixing. Each mixture was then heated to 70�C, stirredvigorously (2200 rpm, 3min) with a mechanical stirrer,vat-pasteurised at 80�C for 10min (Wacher-Rodarteet al., 1993), and cooled down to 45�C. At this point, allthe mixes were inoculated with 0.03 gL�1 lactic culture(MY800, Streptococcus thermophilus and Lactobacillus

delbrueckii subsp. bulgaricus, Industrias Cuamex, S.A.de C.V., Mexico) (Sandoval-Castilla et al., 2004), and

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Table 1

Chemical composition of yoghurts

Yoghurt Total solids

(7SD) (%)

Protein

(7SD) (%)

Fat (7SD)

(%)

FFY 16.47 70.2a 3.42 7 0.3a 2.99 7 0.0b

WPCY 16.907 0.1ab 4.37 7 0.2b 1.45 7 0.0a

MWPY 17.57 7 0.3c 4.52 7 0.2b 1.45 7 0.1a

WPC–MWPY 17.20 7 0.4bc 4.37 7 0.2b 1.45 7 0.0a

FFY: full-fat yoghurt; WPCY: whey protein concentrate yoghurt;

MWPY: microparticulated whey protein yoghurt;

WPC–MWPY blend of whey protein concentrate and microparticu-

lated whey protein yoghurt. Means are the result of nine measure-

ments. Means in a column followed by different letters are significantly

different (pp0.05).

Table 2

Flow rheological parameters of yoghurts

Yoghurt Z0 (7SD) (kPa s) l (7SD) (s) p (7SD)

FFY 76.2 7 6.7c 2435.8 7 189.9bc 0.946 7 0.004c

WPCY 58.7 7 0.6b 1880.4 7 19.1a 0.945 7 0.004c

MWPY 35.1 7 1.0a 2379.6 7 73.6b 0.913 7 0.013b

WPC–MWPY 42.1 7 0.3a 2682.2 7 20.6c 0.889 7 0.003a

FFY: full-fat yoghurt; WPCY: whey protein concentrate yoghurt;

MWPY: microparticulated whey protein yoghurt;

WPC–MWPY blend of whey protein concentrate and microparticu-

lated whey protein yoghurt. Means are the result of nine measure-

ments. Means in a column followed by different letters are significantly

different (pp0.05). Z0: low shear limiting viscosity; l: time constant

associated to relaxation time; p: shear thinning index.

C. Lobato-Calleros et al. / International Dairy Journal 14 (2004) 777–782 779

incubated at 45�C until an acidity of 90–95�d wasreached. Yoghurt was cooled down and stored at 4�C.Yoghurt samples were withdrawn after 5 days of storagefor rheological evaluation. All treatments were done intriplicate using a completely random experimentaldesign. Chemical composition of the yoghurts is shownin Table 1, and has been reported elsewhere (Sandoval-Castilla et al., 2004).

2.4. Rheological determination

The rheological measurements were performed with aPhysica DSR 4000 Dynamic Shear Rheometer (PhysicaMesstechnik, Stuttgart, Germany), with a cone-plategeometry, in which the rotating cone was 75mm indiameter, and cone angle of 2�. Temperature main-tenance was achieved with Physica TEK 150P tempera-ture control and measuring system. Yoghurt sampleswere carefully placed in the measuring system, and leftto rest for 1 h for structure recovery and temperatureequilibration. All the measurements were carried out at5�C.

Flow curves of the yoghurts were obtained by varyingthe shear rate from 10�4 to 103 s�1 in 24 steps with equaldistance in a logarithmic scale, and the correspondingshear stress values measured. The flow curves data wasfitted using the rheometer software to the Carreau,Casson, Cross, Herschel-Bulkley, and Ellis rheologicalmodels (Steffe, 1996) that describe the behaviour oftime-independent fluids.

In the creep compliance test the undeformed samplesof yoghurts were suddenly subjected to a constant shearstress of 9 Pa (constant torque of 1mNm). Thedeformation of the viscoelastic materials increases withtime and approaches a steady state where the deforma-tion rate remains constant, in this point the stress can besuddenly removed and can be analysed for recoverableshear (Rao et al., 1987). All the yoghurt treatmentsreached a constant deformation rate within 300 s, and atthis time the applied stress was released to allow sample

recovery. Only the creep compliance region of the curveswas analised in this work. The applied stress fell withinthe linear viscoelastic region of the yoghurts. Determi-nation of the linear viscoelastic region was done byperforming stress and frequency sweep tests.

2.5. Statistical analyses

An analysis of variance (ANOVA) and Tukey’s test(ap0.05) were performed on rheological data of theyoghurts using the Statgraphics 7 statistical analysissystem (Statistical Graphics Corp. Manugistics Inc.,Cambridge, MA).

3. Results and discussion

3.1. Flow properties

The model that best fitted the log viscosity versus logshear rate data of all the yoghurts was the Ellis equation(R2>0.99) (Darby, 1996):

Z ¼Z0

1þ ðl’gÞ2� �p ð1Þ

where Z is the apparent viscosity, Z0 is a low shearlimiting viscosity, l is a time constant associated to therelaxation time of polymers in solution, and p is a shearthinning index. The values of the Ellis equationrheological parameters for the yoghurts are given inTable 2. Foods described by Ellis model possess a‘‘structural viscosity’’, characterised by Newtonian flowat very low shear rates (Z0), attributed to the formationof a reversible ‘‘structure’’ or network in the ‘‘rest’’ state(Darby, 1996), and displayed by foods containingmacromolecules, which under the influence of shear,commence to stretch-out and deform exhibiting acharacteristic relaxation time (Steffe, 1996). When thebiopolymer molecules become aligned with the field ofshear and weak physical interactions responsible forbiopolymer–biopolymer interactions are disrupted their

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Table 3

Viscoelastic parameters of yoghurts

Yoghurt J0 (7SD) (mPa�1) Jm (7SD) (mPa�1) im (7SD) (s) ZN (7SD) (kPa s)

FFY 2.14 7 0.3a 2.11 7 0.4a 36.62 7 4.4c 133.02 7 13.2b

WPCY 1.84 7 0.1a 2.08 7 0.1a 28.55 7 0.2a 120.19 7 7.1b

MWPY 4.13 7 0.3b 5.22 7 0.5b 33.17 7 2.2bc 54.77 7 8.2a

WPC–MWPY 3.71 7 0.2b 4.57 7 0.1b 33.30 7 1.4bc 60.79 7 3.2a

FFY: full-fat yoghurt; WPCY: whey protein concentrate yoghurt;

MWPY: microparticulated whey protein yoghurt;

WPC–MWPY blend of whey protein concentrate and microparticulated whey protein yoghurt. Means are the result of nine measurements. Means in

a column followed by different letters are significantly different (pp0.05). J0: instantaneous compliance; Jm: mean compliance; im: mean retardation

time; ZN: Newtonian viscosity.

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apparent viscosity decreases, with the food oftenexhibiting strong shear-thinning behaviour at intermedi-ate shear rates (McClements, 1999). l provides an orderof the critical shear rate marking the end of the zeroshear rate Newtonian region and the onset of the shearthinning region, and p tends to a value of (1�n), where n

is the power-law flow behaviour index (Rao, 1999). Asthe value of l was significantly different (pp0.5) for theyoghurts made with WPC, MWP and the blend ofWPC-MWP, it can be inferred that these fat replacerscontributed differently to yoghurt casein networkstructure. However, the WPCY showed non-significantdifference with the FFY (p>0.05), but both showedsignificantly higher p values (lower n values) than theMWPY and the WPC-MWPY (pp0.05). Shear thinningbehaviour is closely associated to mouthfeel. Liquidfoods not exhibiting extensive shear thinning behaviourat the shear stresses experienced within the mouth areperceived as being ‘‘slimy’’, but a certain amount ofviscosity is needed to contribute to the ‘‘creaminess’’ of aproduct (McClements, 1999). In this regard, we assumethat the WPC fat replacer was the one that providedreduced-fat yoghurt with a mouthfeel resembling moreclosely that of the FFY, and the WPCY was the oneshowing the closest Z0 value to that of the FFY.

3.2. Creep compliance properties

All of the yoghurts exhibited typical creep compliance(J)-time (t) curves (Curves not shown), from which theviscoelastic parameters were calculated in accordancewith the relationship (Sherman, 1970):

JðtÞ ¼ J0 þX

i

Jm½1� expð�t=tmÞ þ t=ZN; ð2Þ

where J0(=1/E0) is the instantaneous complianceobtained when the bonds between the structural unitsare stretched elastically, E0 is the instantaneous elasticmodulus; Jm is the mean compliance related with thephase of the test during which the bonds break andreform, but all of them do not break and reform at thesame rate; tm is the mean retardation time i.e. the timetaken for the delayed deformation to reach approxi-

mately 63.2% (1�1/e) of the final value, and ZN is theNewtonian viscosity associated with flow of thestructural units of the sample food as result of apronounced rupture of bonds (Rao et al., 1987). Thevalues of these parameters for the different yoghurts areshown in Table 3.

WPC, MWP and the blend of both whey proteinsaffected the creep compliance behaviour of reduced-fatyoghurt in different ways depending of their structuraland functional properties. WPC provided to the yoghurtsimilar values of J0 than those exhibited by the FFY(p>0.05), i.e. both yoghurts had resembling elasticity.In full-fat set yoghurt the main milk proteins, caseins,form an uninterrupted network composed of chains andclusters of casein micelles (Kalab, 1979), in which fatglobules can interact with the gel casein matrix asbinders providing a strong elastic structure (Lucey et al.,1998).

Addition of small amounts of whey protein to skimmilk gels reinforces the structure, probably forming asecondary network in the interstices left by casein chains(Aguilera & Kinsella, 1991). Sandoval-Castilla et al.(2004) reported that WPC incorporation to reduced-fatyoghurt resulted in a protein network formed mainly bycasein micelle chains (protein particles linked in chains)rather than clusters (protein particles held together togreater extent forming relatively large aggregates) withsmall spaces originally occupied by whey, whichexhibited firmness, tension and springiness comparablewith those of FFY. Puvanenthiran, Williams, andAugustin (2002) observed that as the casein to wheyprotein ratio was decreased, by blending skim milk withWPC, the maximum gel strength of the yoghurtincreased. Whey protein induced a finer proteinstructure with numerous small pores and a densenetwork of crosslinks.

The addition of MWP alone or combined with WPCyielded yoghurts with significantly higher J0 values(pp0.05) than that of the FFY and WPCY. Sincecompliance is the strain per unit stress, a higher instantcompliance indicates a greater degree of deformationand lower recovery ability. Sandoval-Castilla et al.(2004) reported that the corpuscular nature of MWP

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was maintained when forming part of the protein matrixof reduced-fat yoghurt, causing a more open proteinstructure in comparison with that showed by FFY, aswhey protein particles interrupted the casein micellesclusters. These same authors stated that when using ablend of WPC–MWP the reduced-fat yoghurt proteinnetwork was formed by predominantly casein micelleschains, but some of them were fused into clusters, withparticles of MWP abutting from the latter. These resultsindicate that the role played by the MWP particles in thereinforcement of yoghurt gels was relatively less thanthat by WPC. Partial heat denaturation of WPCproduces a majority of soluble aggregated proteins,favouring their interaction with other proteins i.e. withk-casein on the surface of casein micelles via disulphidebonding (Beuschel, Culbertson, Partridge, & Smith,1992; Pfizer Food Science Group, 1995). This effectivelyincreases the concentration of gel-forming protein in theyoghurt matrix (Keogh & O’Kennedy, 1998).

im may be considered a measure of the complexity ofthe type and diversity of the bonds in the structure(Lobato-Calleros, Aguirre-Mandujano, Vernon-Carter,& S!anchez-Garc!ıa, 2000). The retardation time is uniquefor each material, and in viscoelastic materials, the timeto achieve maximum deformation is delayed. Materialswith large retardation times reach full deformationslowly (Steffe, 1996). The value of im was significantlydifferent (pp0.05) between FFY and WPCY, but nosignificant difference existed (p>0.05) among the FFY,MWPY and WPC–MWPY. Bond rupture rate inyoghurts is apparently affected by the relative amountof chains or clusters of casein micelles in the proteinnetwork. When the clusters of casein micelles predomi-nate in the protein network bond deformation proceedsmore slowly and retardation times are longer than whencasein chains predominate. SEM micrographs reportedby Sandoval-Castilla et al. (2004) showed that theprotein network of the FFY was composed by a greaternumber of clusters of casein micelles than of caseinmicelles chains. MWPY showed a similar structure tothat of the FFY but more open and less dense, probablydue to the interrupted protein network caused by MWPparticles and to the lower number of fat globules actingas linking protein agents. In contrast, in the WPCY theprotein network was mainly made-up by casein micelleslinked by particle-to-particle attachment in long chains.The WPC–MWPY showed a protein network resem-bling that of MWPY but with casein micelles chainsrather than clusters, and with a more compact and lessopen structure.

The value of Jm was non-significantly differentbetween the FFY and WPCY yoghurts (p>0.05) butboth showed significantly lower Jm values (pp0.05)compared to those of the MWP and WPC-MWPY. Jm isthe ratio between the mean viscosity (Zm) and theretarded elasticity (Em), so that high Jm values indicate a

predominating viscous nature of the network ratherthan an elastic nature. Renard, Robert, Faucheron, andSanchez (1999) reported that increasing MWP concen-tration in mixed gels made of MWP and b-lactoglobulin,led to heterogeneity in the protein network (due to sterichindrance or to segregative thermodynamic incompat-ibility) with a decrease in the elastic part and an increasein the viscous part in the network.

WPCY and FFY showed non-significantly different(p>0.05) ZN values. Following the rupture of some ofthe bonds, the time required for them to reform is longerthan the test period, so that the broken bonds releasestructure units that flow past one another (Sherman,1970). Both in FFY and in WPCY, the casein micellesand/or whey proteins making-up the gel structure haverelatively small sizes (Puvanenthiran et al., 2002), so thatwhen structure rupture occurs, the number of releasedunits is very high, causing distortion of the flow pathwayas these ‘‘chunks of structure’’ flow past one another,causing an increase in the Newtonian viscosity. Tamimeet al. (1995) reported that MWP molecules were notfreely dispersed into the aqueous phase maintainingtheir corpuscular nature in yoghurt made from skimmilk, so that when structure rupture occurs, the numberof released units is relatively lower in the MWPY andWPC–MWPY, showing lower ZN values than the FFYand WPCY.

4. Conclusions

The flow and creep compliance data were useful forexplaining the effect of WPC, MWP, and their blend onthe protein network of reduced-fat yoghurts. Therheological data indicate that the different fat replacersprovided different effects in improving the proteinnetwork of reduced-fat yoghurt. Added WPC resultedin a protein network structure showing flow andviscoelastic properties similar to those exhibited by theFFY. The incorporation of MWP alone or combinedwith WPC did not improve the rheological properties ofreduced-fat yoghurt.

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