Critical Reviews in Food Science and Nutrition, 49:153–163 (2009)Copyright C©© Taylor and Francis Group, LLCISSN: 1040-8398DOI: 10.1080/10408390701764807

Application of Advanced InstrumentalMethods for Yogurt Analysis

AMIR M. MORTAZAVIAN,1 KARAMATOLLAH REZAEI2 andSARA SOHRABVANDI1

1Department of Food Technology, Faculty of Nutritional Sciences and Food Technology/National Nutrition and FoodTechnology Research Institute, Shahid Beheshti University (MC), P.O. Box 19395–4741, Tehran, Iran2Department of Food Science, Engineering and Technology, Faculty of Biosystem Engineering, University of Tehran,31587-77871, Karaj, Iran

Compared to the classical methods of analysis, advanced instrumental methods have received increasing attention due totheir highly precise analysis of food micro-/macro-structure. Due to its widespread popularity, yogurt has been the subject ofnumerous studies. This article discusses major advanced instrumental methods applied to the analysis of set/stirred yogurtreported in the literature. Discussed analytical methods have been categorized into two parts, namely chemical analysismethods (including flavor analysis of yogurt, analysis of milk constituents, and assays of indexes), and structural analysismethods (including textural and rheological analysis as well as microstructural analysis).

Keywords Flavor, GC, HPLC, microstructure, rheology, texture, viscometry, viscosity, yogurt

INTRODUCTION

Analysis of the qualitative parameters of yogurt can be di-vided into two parts as chemical and structural analysis. Theformer includes yogurt flavor analysis (taste/aroma compounds),milk constituents analysis (e.g., carbohydrates, fat, total nitro-gen, free amino acids, proteins, and antibiotics), and assays forchemical indices (e.g., Zeta potential of casein micelles andenzymes activity). Structural analysis includes textural and rhe-ological as well as microstructural analysis. Textural and rhe-ological analysis is associated with the methods evaluating gel(set-type yogurt) and liquid (stirred-type yogurt) properties ofyogurts. Microstructural analysis consists of two parts: mi-crostructural images (studying detail structure) and microstruc-tural assays (such as those obtained by EPS, exopolysaccaridessecreted by bacteria, and those for the determination of the meandiameter of fat globules/particles).

From the characteristics mentioned above, flavor consists ofthose attributes in the food that are perceived by two senses astaste and smell (de Man, 1999). In fact, typical flavor of a productexhibits part of its identity and therefore, flavor analysis of foodsis highly important since it results in the determination of their

Address correspondence to Dr. Karamatollah Rezaei, Department ofFood Science, Engineering and Technology, Faculty of Biosystem Engineer-ing,University of Tehran, 31587-77871, Karaj, Iran. E-mail: krezaee@ut.ac.ir

odor profiles, which could be utilized for both quality controland research developments. Flavor analysis of yogurt has beenthe subject of numerous research in the area of dairy science.Such importance arises from the fact that flavor is vital part ofyogurt’s critical value.

From the taste point of view, lactic acid has been found asa key taste component in yogurt. However, other organic acidsand some additives are also involved in the taste of this product(Tamime and Robinson, 1999). It should be pointed out that thedegree of sourness in yogurt strongly depends upon the con-sumers’ perception and their eating habits; however, excessiveacidity results in off-flavor anyways. In general, compared to theanalysis of nonvolatile compounds and texture analysis, odoranalysis is rather complicated, which can be attributed to thevast range of volatile components at very low concentrations.Also, there are many interactions (synergistic or antagonistic)between different volatiles and volatiles associated with suchparameters as acidity or pH, which considerably influence thearoma perception (Ott et al., 2000a). In addition, some volatilecomponents, known as key odor compounds, potentially havestronger contribution in the perception than do the others. Con-sequently, the odor profile of a product may not directly be as-sociated with what is perceived by olfactory senses. Accordingto many studies (Kneifel et al., 1992; Chandan and Shahani,1993; Imhof et al., 1995; Tamime and Robinson, 1999), car-bonyl compounds such as acetaldehyde (in particular), diacetyl,

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acetoin, and acetone are by far the most important compo-nents responsible for characteristic/typical aroma of plain yo-gurt. These volatiles are substantially produced during the fer-mentation stage (Kwak et al., 1996; Stanley, 1998; Tamime andRobinson, 1999). However, such understanding is not final sinceit fails to explain many fine details and their causes (Mortazavianand Sohrabvandi, 2004).

As part of other chemical analysis parameters, chemical in-dices such as Zeta potential of casein micelles and enzymaticactivity of bacterial starter cultures are important parameters inorder to monitor the aggregation/fusion potential during gelation(Mottar et al., 1989) and the formation rates of casein micellesand casein particles during fermentation/incubation time.

Textural and rheological properties of yogurt (gel elasticity,firmness, and cohesiveness of set yogurt, and apparent viscos-ity and viscoelasticity of stirred yogurt), which have been thesubject of many investigations, also have great influence on con-sumer perception. Set yogurt exhibits gel properties (viscoelas-tic) with relatively small yield value. Rheological behaviors ofthis product are normally expressed by “consistency,” “firm-ness,” and “hardness”. Stirring of set yogurt results in the degra-dation of the tridimentional gel network and as a consequence, inthe formation of stirred yogurt, which is also a viscoelastic mate-rial. Compared to set yogurt, stirred yogurt is more viscous thanelastic at higher shear rates. Therefore, the rheological propertiesof stirred yogurt are normally assessed by measuring apparentviscosity (Skriver et al., 1999; Walstra et al., 1999). Stirred yo-gurt has both pseudoplastic (shear thining) and thixotropic (sheartickening) characteristics (Prentice, 1992). The aim of rheolog-ical assessments is to quantify the textural parameters in orderto approach a global scientific language for quality control ofproducts and also for their research developments. Benezech andMaingonnat (1992; 1993; 1994) have reviewed the rheologicalcharacteristics and flow properties of yogurt.

Microstructure of food materials is caused by the type, num-ber, and arrangements of their micro-/macro-molecules and alsoby the interactions occurring among them. Microstructure gen-erates the texture as well as the appearance, which are part ofmacrostructure. Microstructure studies give the possibility tolearn about and justify the cause of numerous textural prop-erties and rheological behaviors leading to the knowledge ofimproving, maintaining, and designing the texture. Accuracyof rheological properties and behaviors can be more profoundwhen supported by microstructural justifications and elabora-tions. Microstructural elements of set and stirred yogurts andtheir correlations with yogurt rheological properties have beendiscussed by Mortazavian and Sohrabvandi (2004) based on theproposed proportions and moduli. Microstructural analysis canbe performed by studying detailed structural (microscopic im-ages) and/or chemo-structural determinations. In this article,the former has been applied as “microstructural image analy-sis” and the latter as “microstructural assays.” The most impor-tant microstructural assessments related to yogurt are discussedhere.

CHEMICAL ANALYSIS

Flavor Analysis

Gas Chromatography

Among the advanced methods used for the identification andquantification of volatiles in yogurt, gas chromatography (GC)has been the most efficient one (Brauss et al., 1999; Kneifel etal., 1992; Laye et al., 1993; Imhof and Bosset, 1994; Imhof et al.,1994 and 1995; Kwak et al., 1996; Kang et al., 1988; Gardini etal., 1999; Ott et al., 1997; 1999 2000a,b). Ott and his coworkers(Ott et al., 1997; 1999; 2000a,b) are among many researcherswho have contributed to the studies performed on this area. Theyapplied combined static and dynamic headspace GC-MS andpreparative simultaneous distillation-extraction under vacuumin order to separate and identify the volatile components in plainyogurt and were able to identify 91 odorants, 21 of which had keyeffects on the aroma of yogurt. Ott et al. (1997; 1999; 2000a,b)were the first to identify intense flavor components in yogurt,namely 1-nonen-3-one, methional, 2E-nonenal, guaiacol, and 2-mehtyltetrahydrothiophen-3-one. 1-nonen-3-one, generated byheat oxidation, was found as a very potent odorant with the low-est realized odor threshold (8 pg/kg). Prior to findings by Ott et al.(1997), only about 60 volatiles had been identified from yogurt(Lay et al., 1993; Imhof and Bosset, 1994; Imhof et al., 1994;1995). Similar to many other methods, GC methods consist oftwo separate stages including a sampling step and the instru-mental analysis (identification/determination). Yogurt does nothave an intense/sharp odor characteristic and therefore, it is cat-egorized as a product with a mild aroma (Ott et al., 1997). As aresult, a preheating stage is necessary for sampling purposes inorder to increase the volatility of aroma. This in turn might leadto a change in the aroma profile of yogurt. Therefore, mild sam-pling techniques such as purge-and-trap are suggested (Ott et al.,1997). GC-olfactometric techniques (also known as hyphenatedGC-O) are based on the dilution sniffing (also known as sniffingmethod) letting direct identification of aroma compounds. Di-lution sniffing is carried out in a sniffing port and is continueduntil no volatiles could be detected. Charm analysis (Acree et al.,1984) and aroma extract dilution analysis (AEDA) (Ullrich andGrosch, 1987) are two common methods of olfactometry. Kwaket al. (1996) used a headspace GC (HS-GC) sampling methodbased on the procedure reported by Bassette and Ward (1975) toanalyze the volatile components of yogurt. Chen et al. (1998),based on a procedure proposed by Narain et al. (1990), applieddynamic headspace GC-MS and purge and trap injectors to as-sess the effect of adding yeast species on yogurt flavor profile.Linssen et al. (1991) utilized the dynamic headspace GC-MSmethod to investigate the adsorption of different componentsof drinking yogurt on the high-density polyethylene (HDPE)packaging material. Also, Kneifel et al. (1992) studied the flavorprofile analysis of yogurt using HSGC according to the proce-dure reported by Ulberth (1991). Ott et al. (1999) utilized quick

APPLICATION OF ADVANCED INSTRUMENTAL METHODS FOR YOGURT ANALYSIS 155

HS-GC to quantify vicinal diketones (such as 2,3-butandioneand 2,3-pentandione) in acidic or mildly acidic yogurts. Utiliz-ing continuous monitoring by atmospheric pressure ionization-mass spectrometry (API-MS) using plate-form quadruple MS,Brauss et al. (1999) investigated the effect of fat content on thevolatile composition, volatile intensity, and the rate of volatilerelease. Gardini et al. (1999) studied the effect of culture com-position containing Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus salivarius ssp. thermophilus, and Lactobacillusacidophilus on the aroma profiles of fermented milks using theGC method. By applying a GC-olfactometry method, Bendallet al. (2001) detected more than 70 odorants from fresh milk andreported that hept-cis-4-enal was one of the key components offresh milk.

High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) has beenwidely used for the determination of organic acids such as lac-tic, acetic, pyruvic, uric, formic, propionic, butyric, and hippuricacids (Marsili et al., 1981; Fernandez-Garcia and McGregor,1994; Barrantes et al., 1996a; Fernandez-Garcia et al., 1998;Beal et al., 1999; Adhikari et al., 2000; Lee et al., 2001). Whenusing HPLC, total acids are normally extracted with sulfuricacid and then separated on an ion-exchange column. Quantifi-cation is commonly carried out by utilizing external standards(Fernandez-Garcia and McGregor, 1994; Fernandez-Garcia etal., 1998; Beal et al., 1999). In order to avoid any interferences,it is necessary that proteins be precipitated before analysis. Todo this, trichloroacetic acid treatment followed by centrifuga-tion has successfully been applied (Beal et al., 1999). Adhikariet al. (2000) investigated the effect of microencapsulation of bi-fidobacteria on the concentrations of produced lactic and aceticacids in yogurt using ion-exchange HPLC based on modifiedmethods of Bouzas et al. (1991) and Gonzales de Llano et al.(1996). Kwak et al. (1996), according to the Hodgin et al. (1983)procedure, used the HPLC method for studying the kinetics offree amino acids production during the fermentation. Such studywas performed to monitor the increase in the bitterness in thefermented milks.

Electronic Nose Methodology

Flavor release profiles throughout the fermentation and ag-ing of food materials can be measured using electronic noses(e-noses). The electronic nose is a device composed of an arrayof gas sensors with nonspecific responses that has pattern recog-nition ability using multivariate data analysis. The informationfrom the sensors are collected through such pattern recognitiontechniques as principal component analysis (PCA) or artificialneural networks (ANN). This methodology, which is a promis-ing approach for real-time, in-line, in situ determinations andnondestructive sensing, is highly useful for the food industry(Mong et al., 2004). Recently, this method has been successfullyapplied for the analysis of various food materials (O’Connellet al. 2001; Mong et al., 2004) and it can also be used for yogurtanalysis.

Analysis of Milk Constituents

Total nitrogen corresponding to the Kjeldahl method couldalso be determined using such instruments as the Gerhard diges-tion machine (Hess et al., 1997). Mono- and disaccharides canbe analyzed using an HPLC system, after the precipitation ofproteins with thrichloroacetic acid (Bouzar et al., 1997). Also,gas-liquid chromatography (GLC) has efficiently been used formonosaccharide quantification (Blakeney, 1983). The above-mentioned methods could be a replacement for classical methodsof polarimetry in the case of lactose determination (Biggs andSzijarto, 1963). Smitinont et al. (1999) determined monosac-charides by HPLC equipped with Phenomenex Spherisorb-NH2

column and a refractive index detector. Fatty acid compositionof yogurt has been determined using GLC on the lipid extractedby the Rose-Gottlieb method according to British Standards In-stitute (BSI, 1980; Barrantes et al., 1996a). Beal et al. (1999)determined the concentrations of lactose, galactose, and totallactic acid of fermented milks using cation exchange HPLC af-ter precipitating proteins of the samples with trichloroacetic acidfollowed by centrifugation. HPLC has also been applied for thequantification of vitamins A and D (Faulkner et al., 2000). Leeet al. (2001) invented a simple and fast method for the identi-fication of bifidobacteria, which is found in fermented milks,using thin layer chromatography (TLC) based on the producedshort-chain fatty acids (SCFA) including lactic, acetic, propi-onic, succinic, citric, and butyric acids in the culture broth.The method was founded upon the different colors exhibitedafter spraying of the chromatogram with the indicator solution(methylred-bromophenol blue in 70% ethanol).

Analysis of Chemical Indices

Zeta potential (ζ ) of casein micelles has been measured usingMalvern Zetasizer 11 based on an electrophoresis method andlaser light scattering detection (Mottar et al., 1989). Also, theenzymatic activities of bacterial starter cultures have been mea-sured using o-pthaldialdehyde-based spectrophotometric assay(OPA method). The applied enzymes were amino-, di-, tri-, andendopeptidase (Shihata and Shah, 2000). The OPA method wasused for the first time when it was introduced by Church et al.(1983) in order to assess the concentrations of free amino groupsin the filtrate.

STRUCTURAL ANALYSIS

Textural and Rheological Analysis

Penetration and Texture Profile Analysis (TPA) Tests

In a penetration test (also called puncture test when the probepunches the sample surface), a cylindrical plunger/spindle/probe(flat-based or cone-shaped in the viewpoint of probe configura-tion) with a defined diameter (mm), the penetration speed/rate

156 A. M. MORTAZAVIAN ET AL.

Table 1 Selected publications applying penetration and TPA tests

Source Method InstrumentRecorded

Parameter(s)

Mohamed and Morris(1987)

TPA Instron Universal testingmachine

Firmness

Mottar et al. (1989) Penetration Instron food testing machine HardnessTamime et al. (1991a,b) Penetration Stevens-LFRA texture

analyzerFirmness

Fiszman et al. (1999) Penetration TP-XT2texture analyzer Firmness value and force atbreak (gel rigidity index)

Martinou-Voulasiki andZerfiridis (1990)

Penetration Penetrometer Firmness

Barrantes et al. (1996b) Penetration Stevens-LFRA textureanalyzer

Firmness

Hassan et al. (1996b) TPA Instron Universal testingmachine

Yield stress and firmness

Tamime et al. (1996) Penetration Stevens-LFRA textureanalyzer

Firmness

Hess et al. (1997) Penetration TP-XT2texture analyzer Gel strengthRawson and Marshall

(1997)Penetration and

combination of backextrusion and TPA tastes

TA-XT2texture analyzer Adhesiveness, springiness,cohesiveness, andhardness (for stirredyogurt)

Suwonsichon and Pelegy(1999)

Compression TA-XT2texture analyzer Firmness (force versusdistance curves) and TPAanalysis

Fertsch et al. (2002) Penetration TA-XT2texture analyzer FirmnessPuvanenthiran and

Auhustin (2002)TPA Instron Universal testing

machineGel strength, force at yield,

initial gradient and stressrelaxation

(mm/s), the penetration depth and weight, and the experimen-tal temperature forces the surface of foodstuff and penetratesinto them. As a result, the penetration curve (penetration forceversus penetration depth) is recorded. In penetration tests, sev-eral parameters including force at break (hardness, N) or thedistance at which the breaking takes place (consistency, mm),the mean slope of force-distance or penetration curve (gel rigid-ity index, N/mm), the maximum force required for the probeto penetrate the gel (gel strength, N), and the penetration depthwithin the defined time (firmness) can be measured. For sam-ples not broken at a defined compression force, the penetrationforce (N) at a determined displacement can be recorded as firm-ness (Martinou-Voulasiki and Zerfiridis, 1990; Hess et al., 1997;Fiszman and Salvador, 1999). If the probe deforms the surfacewithout penetration (due to the high surface area of the probe orlow penetration force), the test is a so called “compression test”(Steff, 1996a).

The texture profile analysis (TPA) test is a fairly popu-lar one where a double compression force is applied to thesample and consequently the rheological parameters includ-ing fracture/fracturability, hardness, cohesiveness, adhesive-ness, springiness, gumminess, and chewiness are measured(Tunick, 2000). TPA tests and their parameters have previouslybeen discussed by Tunick (2000) as well as by Mortazavian andSohrabvandi (2004). In TPA assessment, the curve of force ver-sus time is recorded. TPA is performed by sending a crossheaddown a vertical column, causing a flat plate to deform a cylindri-

cal specimen placed on a lower plate. The crosshead then returnsat the same rate and repeats the procedure, roughly mimickingtwo biting actions on a piece of sample. This test is highly usefulfor obtaining a general indication of texture and for making com-parisons among several samples (Tunick, 2000). Contrary to thedynamic-progressive profiling methods, TPA does not provideadequate information about the dynamic features of the samples(Wilkinson et al., 2000). Several studies in which penetrationand TPA tests have been applied are listed in Table 1.

Viscometric Tests

In these tests, different types of viscometers are used tomeasure the viscosity of stirred yogurt. In the case of set yo-gurt, an arbitrary procedure such as stirring the gel with aglass rod, 10 times (or more) clockwise and 10 times (ormore) anticlockwise can be adopted to break and stir the gelin order to evaluate the fractured gel viscosity (Rawson andMarshall, 1997). Two types of viscosities can be measured:shear-free/elongational/extentional viscosity and shear viscos-ity. In shear-free viscosity, there is no velocity gradient amongliquid molecules when the liquid starts flowing, which can be as-sessed through different procedures discussed by Steff (1996b).Shear viscosity of yogurt is commonly assessed using rota-tional viscometers (Rhom, 1992; Rhom and Kovac, 1995; Has-san et al., 1996a; Bouzar et al., 1997; Skriver et al., 1999).

APPLICATION OF ADVANCED INSTRUMENTAL METHODS FOR YOGURT ANALYSIS 157

Rotational viscometers are very sensitive, can operate at lowshear rates and also can take precise measurements of viscosity(Cullen et al., 2000). The viscosity measured by using a rota-tional viscometer is the so-called “rotational viscosity” (Rawsonand Marshall, 1997; Bouzar et al., 1997). Since stirred yogurtis a non-Newtonian fluid, its shear viscosity is reported by theword “apparent viscosity or η.” Apparent viscosity of yogurtcan be measured as a function of shear rate or as a function oftime at a fixed shear rate. The latter is so-called “steady-shearviscosity.” The curves of the apparent viscosity versus the shearrate indicate pseudoplastic or dillatant property and those of theapparent viscosity versus time represent thixotropic/shear thin-ning or rheopectic/shear thickening property (Steff, 1996a). Inpseudoplastic curves, η′ decreases with an increase in shear rate.While, in dillatant curves, changes in η′ is opposite. The curvesof η′ versus time provide information about the time dependentproperties of the samples. Decreased and increased apparentviscosities within a given time in a fixed shear rate imply shearthinning and shear thickening behaviors, respectively. If visco-metric forces increase gradually from their lowest levels, theyield stress of stirred yogurt can also be measured (Hassan etal., 1996a; Rawson and Marshall, 1997). A useful viscometryprocedure is to measure the viscosity from extremely low shear

rates (known as zero shear viscosity or η0) up to extremely highshear rates (called as extreme shear viscosity or η∞). Recordingthe shear rate-viscosity curve results in two remarkable breakswithin the curve, where zero and extreme areas are separatedfrom the intermediate area. Yogurt shows almost a Newtonianbehavior at extreme viscosities (Steff, 1996a; Allmere et al.,1998; 1999). In order to measure shear-free/elongational vis-cosity, a simple practice is to pour the stirred yogurt downwardfrom an orifice with a defined diameter as a function of grav-ity acceleration at a fixed temperature and measure the movingdistance during a time period (Steff, 1996b). Some instrumentscan be applied for several different measurements. Table 2 listsseveral published articles on viscometric tests.

Oscillatory Tests

Oscillatory tests (as a transient/dynamic measurement, alsocalled harmonic assessment) have been vastly used to evalu-ate the rheological properties of set and stirred yogurts, cream,cheese, and soft ice cream (Tunik, 2000). They are among thebest practices to measure the viscoelastic properties of gels andgel-like materials, i.e., the materials that exhibit viscoelastic be-havior. In oscillatory tests, materials undergo two simultaneous

Table 2 Selected publications applying viscometric tests

Source Measuring system Recorded parameter

Mottar et al. (1989) Haake Rotovisco RV2 viscometer using a MVI measuringsystem

Apparent viscosity and the magnitude ofstructure breakdown

Martinou-Vulasiki andZerfiridis (1990)

Brookfield viscometer Mix viscosity at yogurt ice cream

Rohm (1992) RFS2 spectrometer equipped with a cone-and-plate system anda ES environmental system connected to a D&GH circulator

Dynamic shear viscosity (η′)

Lorenzi et al. (1995) Rotational coaxial-cylinder viscometer Haake Rotovisko RV100

Continuous shear flow test by recording shearstress versus shear rate

Rohm and Kovak (1995) RFS2 spectrometer equipped with a cone-and-plate system anda ES environmental system connected to a D&GH circulator

Dynamic shear viscosity (η′)

Hassan et al. (1996a) Rotational viscometer equipped with an M5 measuring headand MVI sensor in a concentric cylindrical cup

Shear stress versus shear rate curves (apparentviscosity), consistency coefficient (K), flowbehavior index (n), and strain versusviscosity curves

Bouzar et al. (1997) Rotational viscometer using coaxial cylinder system Apparent viscosityHess et al. (1997) Rheometrics RFS II fluids spectrometer using parallel plate

geometry with constant stressApparent viscosity versus shear rate

Rawson and Marshall (1997) Brookfield Synchro-Lectric RVT viscometer using spindleimmersed to about one third of its length

Apparent viscosity

Fernandez-Gorcia et al. (1998) T-Spindale couple to an LVTD digital viscometer, usinghelipath adaptor thixotropic fluids

Apparent viscosity

Afonso and Maia (1999) TA instruments Weissenberg rheogoniometer, using parallelplate Geometry

Apparent viscosity as a function of shear rateand time

Brauss et al. (1999) RS 150 rheostress Apparent viscositySkriver et al. (1999) Brookfield viscometer Dynamic shear viscosity (η′)Britten and Giroux (2001) Brookfield viscometer mounted on a Helipath support

allowing spindle vertical displacementApparent viscosity

Navarini et al. (2001) Automatic AVS 440 Schott-Geraete equipment with anUbbelohde capillary viscometer immersed in Laudathermostat (Intrinsic viscosity was determined by the doubleextrapolation of both the Huggins and the Kraemer plots).

Relative and intrinsic viscosity as a function ofshear rate

O’Donnell and Butler (2002) Rotational viscometer Initial and equilibrium apparent viscosityO’Donnell and Butler (2002) Tube viscometer Pressure drop-/ flow rate data

158 A. M. MORTAZAVIAN ET AL.

stresses: compression and shear (Steff, 1996c). Recorded pa-rameters are storage/elastic modulus, G′, which shows the elas-tic property of the material; loss modulus, G′′, which indicatesthe viscous property of the material; loss tangent value/modulusor tanδ, which represents the dominant rheological behavior,i.e., viscous/liquid-like or elastic/solid-like behaviors; complexmodulus or G*, which indicates the sum of the viscoelastic prop-erties of the materials; overall resistance of gels to deformationand their shape retention; and finally the dynamic shear viscos-ity, η*, representing the viscoelastic flow properties (Walstra,1984; Dikinson, 1992; Steff, 1996a; Tunik, 2000; Aichinger,2003). The two latter moduli can be used as criteria for evaluat-ing spreadibility of materials (Dikinson, 1992). Whether oscil-latory factors including oscillation frequency are fixed or theyare changing, three types of oscillatory tests can be performedas follows: a frequency-sweep test at a constant strain wave am-plitude and at a given temperature measuring G′ and G′′ againstoscillation frequency, a temperature-sweep test at a constant fre-quency and at a given strain wave amplitude measuring G′ andG′′ against temperature or time, and finally a time-sweep testat a constant frequency and at a given strain wave amplitudeand temperature, measuring G′ and G′′ as a function of time(Allmere et al., 1998; 1999; da Silva and Rao, 1999; de Lorenziet al., 1995; Ozer et al., 1998).

Oscillatory tests offer the possibility of continuous/steadymeasurements of gel viscoelastic properties throughout the gelsetting (structure development rate, SDR) and gel softening(structure destruction/loss rate, SLR) processes. This could bedetermined by crossover of G′ and G′′ curves from each other(Steventon et al., 1990; Biliaderis et al., 1992; Ozer et al., 1998;Afonso and Maia, 1999; da Silva and Rao, 1999). However,unsteady evaluations can also be applied (Ozer et al., 1998).To avoid destructive deformation of the gel structure and main-taining the linear viscoelastic properties, gel evaluation is per-formed by small amplitude oscillatory shear techniques (SAOS)(Dikinson, 1992; da Silva and Rao, 1999; Aichinger et al., 2003).However, large amplitude oscillatory shear measurement hasalso been used for the determination of large deformation proper-ties of acid-milk gels (Lucey and Singh, 1997; Lucey et al., 1997;1999). As a destructive test, constant shear rate measurementshave been applied to measure gel yield (Ronnegard and Dejmek,1993; Lucey, 1999). Dynamic oscillatory shear tests of foods arefully described by Gunasekaran and Ak (2000). Selected publi-cations applying oscillatory tests are listed in Table 3.

Creep Recovery/Creep Relaxation Test

Among the dynamic/transient viscoelastic tests, creep-recovery/relaxation test can also be mentioned. The recordedparameters are the curve of strain as a function of time (forboth loading and unloading operations) and relaxation time.Depending on the behavior of elastic materials against the creep-recovery test, various types of these materials can be recog-nized including time-independent (ideal) elastic materials, time-dependent elastic materials with the yield point, time-dependent

elastic materials without the yield point, and time-dependentelastic materials with plastic/permanent deformation property,with or without yield points (Steff, 1996c; Mortazavian andSohrabvandi, 2004).

Ultrasonic Reflectance and Diffusing Wave Spectroscopy

Ultrasonic reflectance sensors based on the ultrasonic wavereflectance at the liquid interface have been used for assessingdensity and viscosity of liquids and gels (Cullen et al., 2000).Therefore, they can also be adopted for yogurt evaluation. Dif-fusing wave spectroscopy (DWS) or nonevasive dynamic lightscattering technique has also been used to measure the rheolog-ical properties of macromolecular solutions, networks, and gels(Cullen et al., 2000).

MICROSTRUCTURAL ANALYSIS

Microstructural Image Analysis

Scanning electron microscopy (SEM) and transmission elec-tron microscopy (TEM) are the most popular methods to obtainmicrostructural images, in which subjective parameters such asconstituents distributions, arrangements, interactions, and com-positions within the micro-structure, structural uniformity of thetexture, as well as particle size characteristics are evaluated.Microstructural analysis provides the best possible approach tojustify and/or confirm the rheological behaviors of food texture.Nowadays, the word “micro-rheology” is being used for thestudies in which rheological assessments are combined with mi-crostructural analysis. Among the various electron microscopytechniques, cryo-SEM is the most frequently applied one in foodresearch. It is particularly useful for studies on fragile food ma-terials (Wilkinson et al., 2000). SEM image analysis includesseveral stages of sample manipulation such as fixation, dehy-dration, and coating (with a layer of gold or chromium), as wellas the main operation for the sample images (Barrantes et al.,1996b; Aichinger et al., 2003). Despite the dominant usage ofSEM in microstructural studies, it suffers from two limitations.First is that in order to prevent the aggregation effect of chargewithin the nonconductive samples, their surfaces must be coatedby a conductive material (such as chromium or gold) before ex-posure of the sample to electron beam. Secondly, to preventevaporation of water from food texture during the analysis, thesample must be dehydrated or frozen. Both procedures can resultin appreciable changes in the molecular structure of the sample.Compared to SEM, atomic flame microscopic technique (AFM)is more suitable in order to maintain the native structure of thesamples. Therefore, when a more precise microstructural anal-ysis is needed, AFM is a proffered technique (Lent et al., 1998).Moreover, a relatively new technique (environmental scanningelectron microscopy, ESEM) is available which allows samplesto be viewed at any temperature in their natural, fully-hydratedstate. ESEM does not need any special sample preparation.

APPLICATION OF ADVANCED INSTRUMENTAL METHODS FOR YOGURT ANALYSIS 159

Table 3 Selected publications applying oscillatory tests

Source Measuring system Recorded parameter(s)

Bohlin et al. (1984) Bohlin VOR rheometer G′Dejmek et al. (1990) Bohlin VOR rheometer G′ and G′′ as a function of shear strain (γ .)Steventon et al. (1990) Bohlin VOR rheometer G′ and G′′ as a function of shear strain (γ .)Biliaderis et al. (1992) Controlled-stress rheometer Gel development rate (GDR)Rohm (1992) RFS2 spectrometer equipped with a

cone-and-plate system and a ESenvironmental system connected to a D andGH circulator

G′ and G′′

Rohm and Kovac (1995) RFS2 spectrometer equipped with acone-and-plate system and a ESenvironmental system connected to a D andGH circulator

G′, G′′, tanδ, and η* as a function of shearstrain amplitude and angular frequency

Ronnegard and Dejmek (1993) Bohlin VOR rheometer G′; development and break down of yogurtstructure

de Lorenzi et al. (1995) Rotational rheometer Haake Rotovisko R 100(oscillatory shear flow tests: strain sweeptest and frequency sweep test)

G* versus strain curve, G* versusfrequency curve, tanδ versus straincurve, and tanδ versus frequency curve

Hess et al. (1997) Rheometrics RS 11 fluid spectrometer usingparallel plate geometry with constant stress

G′ and G′′ as a function of strain

Lucey et al. (1997) Bohlin VOR rheometer (Low amplitudedynamic oscillation)

Storage modulus (G′) and loss tangent(tanδ)

Lucey and Singh (1997) Bohlin VOR rheometer (Large deformationproperties of the gel)

Storage modulus (G′) and loss tangent(tanδ)

Rawson and Marshall (1997) Brookfield Synchro-Lectric RVT viscometerusing spindle immersed to about one-thirdof its length

Apparent viscosity

Allmere et al. (1998, 1999) Bohlin VOR rheometer G′Lucey et al. (1998a,b) Controlled-stress rheometer (low- and large

amplitude dynamic oscillation)G′ and loss tangent (tanδ); determination of

linear region (LR) of the gelOzer et al. (1998) RheoTech instrumental

controlled-stress-rheometer (amplitudeoscillatory test: discontinues monitoring).Rheometer was set up with parallel ategeometry.

G′, tanδ and G*: Gelation profile of themilk:

Afonso and Maia (1999) TA instruments Weissenberg rheogoniometerusing a parallel plate geometry: steadyoscillatory rheometry (amplitude sweep at aconstant frequency)

GDR by monitoring G′

Allmere et al. (1999) Bohlin VOR rheometer (strain sweep test) G′; determination of linear region (LR) ofthe gel, and development and breakdownof yogurt structure

Fiszman and Salvador (1999) Rheometer Loss of tangent modulusFiszman et al. (1999) Rheometer-low strain amplitude dynamic

oscillationG′ as a function of time at a fixed frequency

and loss tangent value as a function offrequency

Ikeda and Foegeding (1999) Bohlin VOR rheometer Rheological transition which occur duringin situ gel formation

Lucey et al. (1999) Bohlin VOR rheometer (low amplitudedynamic oscillation and large deformationproperties)

Storage modulus (G′) and loss tangent(tanδ)

Skriver et al. (1999) Bohlin VOR rheometer- G′, G′′, G∗, and tanδ

Skriver et al. (1999) Bohlin VOR rheometer with a coaxialmeasuring system

G′, G′′, tanδ, and η*

Navarini et al. (2001) RFS2 8500 spectrometer-steady shearmeasurements

G′, G′′, and G* as a function of frequency

Aichinger et al. (2003) Controlled-stress rheometer, in combinationwith a 4-bladed cruciform vane geometry(oscillatory vane rheometry)

G′

160 A. M. MORTAZAVIAN ET AL.

Table 4 Selected publications related to Scanning electron microscopy (SEM), Transmission electron microscopy (TEM),Cryo-scanning electron microscopy (CSEM), and Confocal scanning laser microscopy (CSLM) techniques

Source Measuring system

Mottar et al. (1989) SEM with sample preparation using microencapsulation techniqueTamime et al. (1991a,b) SEM and TEM: microstructure of Labneh (concentrated yogurt)McMahon et al. (1993) SEM and TEM: microstructure of whey protein systemLucey et al. (1998a) CSLM from the microstructure of acid gels made from heated skim milkFiszman et al. (1999) CSEMOzer et al. (1999) SEM: microstructure of LabnehBikker et al. (2000) CSLM: microstructure of acid gels prepared from heated milk fortified with whey protein mixturesSultana et al. (2000) SEM: scanning electron photomicrograph of alginate-starch microcapsules containing probiotic cells in

yogurtHansen et al. (2002) CSEM: Images from Ca-alginate microspheres containing bifidobacteriaAichinger et al. (2003) TEM: sample fixation and post-fixation with glutaraldehyde and osmium tetroxide/dehydration was

performed in a graded alcohol series (70–100% ethanol) and drying with Polaron critical point drierHarte et al. (2003) TEM: microstructure of the samples subjected to combinations of high hydrostatic pressure and

thermal processing

A major advantage of ESEM is that the influence of environ-mental factors such as relative humidity and gas conditionscan be investigated, also that the effect of mechanical opera-tions can be viewed “live.” Confocal scanning laser microscopy(CSLM) is another useful technique offering the advantage ofa straightforward sample preparation which is also quick andflexible in use. However, the low resolution images obtained

by this technique makes it not admittable for some applica-tions. CSLM is most advantageous in the examination of high-fatfoods, which are difficult to prepare for conventional microscopywithout loss or migration of the fat (Wilkinson et al., 2000).Selected publications on the above-mentioned electron mi-croscopy techniques for the analysis of yogurt have been listed inTable 4.

Table 5 Selected publications on major microstructural assays applicable to yogurt studies

Source Method Application

Mottar et al. (1989) Enzyme-Linked Immunosorbent Assay (ELISA) Determination of the amount of β-lactoglobulin,α-lactalbumin associated with the caseinmicelles

Mottar et al. (1989) Fast protein liquid chromatography (FPLC) / applyingon gel filtration experiment

To judge the state of aggregation of the caseinparticles

Slavik (1994) Luminescence spectroscopy Determination of some molecular detailsMcCrae (1994) and

Barrantes et al. (1996a)Forward Lobe Laser Light Scattering (FLLLS) Fat globule size measurement

Brauss et al. (1999) Laser diffraction method using mastersizer instrument Determination of fat globule particle sizeLakowicz (1999) Luminescence spectroscopy Determination of some molecular detailsMottar et al. (1989) and

Britten and Giroux(2001)

Optical density assessment To judge the state of aggregation of the caseinparticles

Smitinont et al. (1999) Methylation performance & then GC-MS coupled to amass selective detector

EPS microstructure analysis

Smitinont et al. (1999) NMR-H and NMR-C13, by spectrometer equipped with5 mm multinuclear probe

EPS microstructure analysis

Smitinont et al. (1999) High performance size exclusion chromatography(HPSEC)

Molecular weight of EPS

Faber et al. (2001) 1D 1H NMR Spectrum Determination of EPSsstructureHavea et al. (2001) One-dimentional (1D) and two-dimensional (2D)

polyacrylamide gel electrophoresis (PAGE)Characterization and interactions of heat-induced

aggregation of whey protein fractionsNavarini et al. (2001) High-performance size exclusion chromatography

(HP-SEC), with differential refractometer andlow-angle laser light scattering detectors

Weight-average molecular weight of EPS

Navarini et al. (2001) NMR measurement (13C-NMR spectra) Characterization of EPSShaw et al. (2001) X-ray diffraction technique or Fourier-transform

infrared spectroscopyInvestigation of polymeric chain arrangements

and crystallization propertiesLacroix et al. (2002) X-ray diffraction technique or Fourier-transform

infrared spectroscopyInvestigation of polymeric chain arrangements

and crystallization propertiesXu et al. (2004) X-ray diffraction technique or Fourier-transform

infrared spectroscopyInvestigation of polymeric chain arrangements

and crystallization properties

APPLICATION OF ADVANCED INSTRUMENTAL METHODS FOR YOGURT ANALYSIS 161

Microstructural Assay

Microstructural assay indicates the chemo-structural deter-minations of food structure and consists of all structural char-acteristics such as chemical interactions at the molecular level,molecular details of the texture constituents, and molecular sizedeterminations. Microstructural image analysis is not includedas part of these assays. The most important microstructural as-says applicable for yogurt analysis are summarized in Table5. Important microstructural details such as protein chain ar-rangements within the gel structure, characteristics, and averagecoordination of milk proteins and fat droplets in milk disper-sion systems have been investigated using microstructural assaytechniques.

CONCLUSIONS

In the present article, the most important instrumental meth-ods used for different aspects of set and stirred yogurts analysiswere discussed. Advanced instrumental methods such as thoseapplied for flavor analysis, textural, and rheological measure-ments have recently been applied for food analysis due to theircapabilities for profound determinations. New instrumental ap-proaches in food analysis have made the food researchers andfood technologists capable of improving the quality characteris-tics of food products and achieving fundamental rules and prin-ciples in their structure. However, in order to reach a higher effi-ciency in the assessments, future studies should be concentratedon the achieving more precise and more accurate techniques andprocedures with greater simplicity (ease of performance), highersensitivity (lower detection limit), and lower experimental costsby expanding the present techniques or inventing the new ones.

ACKNOWLEDGEMENT

Gratitude is expressed to “Research Council of the Universityof Tehran” and “The Council for Research at the Campus ofAgriculture and Natural Resources of the University of Tehran”for their support.

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