ORIGINAL PAPER

In Situ Ultrasonic Characterization of Cocoa ButterUsing a Chirp

Michaela Häupler & Fernanda Peyronel & Ian Neeson &

Jochen Weiss & Alejandro G. Marangoni

Received: 28 November 2013 /Accepted: 24 January 2014# Springer Science+Business Media New York 2014

Abstract Differences between tempered and untemperedcocoa butter were investigated by an ultrasonic signal “chirp”generated by contact transducers. Polarized light microscopyand powder X-ray diffraction were used to characterize themorphology and polymorphism of tempered and untemperedcocoa butter, whereas pulsed nuclear magnetic resonance wasused to determine the amount of crystalline solids present.Ultrasonic wave velocity and attenuation data were collectedsimultaneously throughout the 5-h crystallization process forcocoa butter. Ultrasonic velocity and attenuation changed atthe different solid fat contents (SFC): 4, 8, and 11 %. Untem-pered cocoa butter showed an attenuation of 3 dB/cm at1.7 MHz and 4 % SFC, whereas tempered cocoa buttershowed an attenuation of 4.5 dB/cm at 1.7 MHz and 4 %SFC. At 3 MHz, the attenuation was 2 dB/cm for untemperedand 6 dB/cm for tempered cocoa butter. Under these condi-tions (4 % SFC, 3 MHz), the chirp wave of tempered sampleshowed a phase angle change of 0.5 rad, whereas the untem-pered sample showed −0.5 rad relative to the canola oil thatwas taken as 0. The study suggests that an ultrasonic chirp canbe effectively used to detect differences between tempered anduntempered cocoa butter when measuring attenuation andultrasonic wave phase angle changes as a function of frequen-cy. The in-line characterization of chocolate “temper” using

such nondestructive ultrasonic measurements could be ap-plied to industrial chocolate manufacturing.

Keywords Cocoa butter . Ultrasound . Chirp . Nondestructive

Introduction

Cocoa butter is the fat phase of chocolate and its crystals areresponsible for the desired mouthfeel, snap, and the gloss of agood quality chocolate (Loisel et al. 1998; Afoakwa et al.2007; Marty and Marangoni 2009). The crystallization ofcocoa butter has been studied by many different authors fromvarious perspectives: polymorphism (Wille and Lutton 1966;Chapman 1971; Van Malssen et al. 1996a; Van Malssen et al.1996b; Loisel et al. 1998; Van Malssen et al. 1999), crystalli-zation kinetics (Padar et al. 2008; Campos et al. 2010), mi-crostructure (Marangoni and McGauley 2003), mechanicalproperties (Stapley et al. 1999; Brunello et al. 2003; Dhonsiand Stapley 2006), origin (Chaiseri and Dimick 1989; Martyand Marangoni 2009), the effects of shear (Bolliger et al.1999; MacMillan et al. 2002; Sonwai and Mackley 2006;Mazzanti et al. 2007; Padar et al. 2009), the influence of thecomposition (Foubert et al. 2004; Van Malssen et al. 1996c),and the ingredients of chocolate (Svanberg et al. 2011). Twotechniques are routinely used to differentiate the polymorphicforms of cocoa butter: powder X-ray diffraction and differen-tial scanning calorimetry (Wille and Lutton 1966; Chapman1971; Timms 1984; Van Malssen et al. 1996a; Van Malssenet al. 1996b; Loisel et al. 1998; Van Malssen et al. 1999;Marangoni and McGauley 2003; Schenk and Peschar 2004;Anihouvi et al. 2013; Acevedo and Marangoni 2014).

It is well known that the best chocolates are structured withcocoa butter with crystals in the βV-polymorphic form. ThisβV form is obtained through a tempering process that follows

M. Häupler : J. WeissDepartment of Food Structure and Functionality, University ofHohenheim, Stuttgart, Germany

F. Peyronel :A. G. Marangoni (*)Department of Food Science, University of Guelph, Guelph, ON,Canadae-mail: amarango@uoguelph.ca

I. NeesonVN Instruments, Elizabethtown, Canada

Food Bioprocess TechnolDOI 10.1007/s11947-014-1273-2

a very specific temperature profile (Stapley et al. 1999;Beckett 2000; Afoakwa et al. 2007; Afoakwa et al. 2009) witha melting range from 32 °C to 34 °C, which is equivalent tothe temperature in the mouth. This results in the pleasantmelting behavior of properly tempered chocolate, where-as a higher melting range results in an unpleasant waxymouthfeel.

The chocolate industry uses a temper meter (Tricor Sys-tems, Elgin, IL, USA) for the detection of the proper “temper”of chocolate (Beckett 2000). The temper meter helps to deter-mine whether a proper chocolate “temper” has been achieved.This is the only method used by the food industry to check thetempering state of chocolate. The machine records the coolingprofile of a chocolate sample. If the sample displays a “flat”temperature-time profile upon crystallization, where the latentheat of crystallization generated is exactly balanced by exter-nal heat sink rate of heat removal, then the chocolate isconsidered to be properly tempered. Excessive temperatureincreases at the crystallization temperature and the absence ofa plateau indicate, respectively, undertempered andovertempered chocolate. This method is sensitive to theamount of crystals present and to the melting point of thosecrystals, which is related to their polymorphic form. However,it does not provide any information about the size of thecrystals present and it is extremely sensitive to sample sizeand viscosity. Moreover, samples need to be collected at amanufacturing site and rapidly transported to the location ofthis tempering unit while crystallization is taking place. Allthese factors will affect the results obtained. Moreover, theprinciple of the analysis is extremely indirect. To be able tojudge the tempering state of chocolate, one needs to know (1)the amount of crystalline mass present (solid fat contents(SFC)), (2) the polymorphic state of the cocoa butter crystalspresent, (3) the melting point of crystals present (related topolymorphism and composition), (4) the size of the crystals.None of these parameters are directly provided by the Tricoranalysis. Conversely, most chocolate manufacturers do notown a pulsed nuclear magnetic resonance (NMR) machinefor SFC determination, a powder X-ray diffraction unit forpolymorphic phase identification, a differential scanning cal-orimeter for melting profile analysis, and a microscope/particle sizer for crystal size analysis. Moreover, the analysisneeds to be conducted rapidly, as the chocolate cannot waitvery long for an analytical result.

Ultrasonic spectrometry is a nondestructive technique,which can be adapted easily to an in-line measurement, whichwould be sensitive to the amount of crystalline mass, crystalsize, and possibly crystal polymorphism (McClements 1991;Saggin and Coupland 2001). Standard pulser ultrasound tech-niques have been used to study fat crystallization by severalauthors (Saggin and Coupland 2002; Sato and Koyano 2004;Singh et al. 2004). However, chirp-based ultrasonics, a tech-nique that exploits the properties of chirp waves (Fig. 1)

(Martini et al. 2005a; Martini et al. 2005b; Martini 2007),has not been yet fully developed or routinely used.

An ultrasonic chirp includes many frequencies and differ-ent amplitudes (Martini et al. 2005a). The advantage of usinga chirp compared with the well-known pulser technique is thenumber of parameters obtained from the chirp (Saggin andCoupland 2002). For example, a 1-MHz chirp was used todetermine the thickness of cheddar and spam (Saggin andCoupland 2001) by using four signals, as the two transducersare put to work as transmitters and receivers. Combining theinformation from all four signals allows the user to discardsignal artifacts inherent to the measurement cell and transduc-ers. Removing these effects is sometimes difficult when asingle channel pulse-echo instrument is used.

Two parameters that are usually obtained from an ultrason-ic experiment are attenuation of the signal (Martini et al.2005b) and velocity of the ultrasonic wave (Singh et al.2004; Dukhin et al. 2005).

In this paper, the attenuation of the ultrasonic wave and thephase angle differences as a function of frequency betweencocoa butter and a reference oil are reported.

The objective of this study was to obtain an ultrasonicspectroscopic signature from a chirp that would indicate thedifferences between in situ tempered and untempered cocoabutter. The analysis of cocoa butter was chosen first in order todecrease the complexity of the system studied. Studies onchocolate will be conducted in the future.

Materials and Methods

Chemical Composition—Fatty Acid Analysis by GasChromatography

The gas chromatography (GC) protocol for the fatty acidmethyl ester analysis was adapted from several references(Christie 1982a; Christie 1982b). Briefly, 10 μL of meltedcocoa butter were added to 4 mL of chloroform:methanolsolution (2:1, v/v; Fisher Scientific). Samples were vortexed

Fig. 1 Screen capture of an ultrasonic chirp signal displayed on the SIA-7

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for 1 min, flushed with nitrogen gas (Boc gases, Guelph, ON),and incubated at 4 °C overnight. On the following day, sam-ples were centrifuged at 1,000 rpm for 10 min at 21 °C toseparate phases. The lower chloroform layer was extractedand transferred to a fresh test tube and dried down with agentle stream of nitrogen gas. The lipid was saponified in0.5 M potassium hydroxide in methanol, vortexed for 1 minand flushed with nitrogen gas. The sample was heated for 1 hat 100 °C with a check every 10 min if enough solvent wasleft. After cooling for 10 min at room temperature, 2 mLhexane (EMD HX0295-1) and 14 % boron trifluoride/methanol (Sigma B1252) were added. Phospholipids wereconverted to fatty acid methyl esters after flushing with nitro-gen gas and an incubation at 100 °C for 1 h. The sample wascooled for 10 min at room temperature before adding 2 mL ofdouble-distilled water to stop the methylation. After spinningat 1,000 rpm for 10 min at 21 °C to separate the phases thehexane layer was extracted into a clean GC vial and driedunder nitrogen.

Fatty acid methyl esters were quantified on an Agilent7890A gas chromatograph equipped with an FID and sepa-rated on an Agilent J &W fused-silica capillary column (DB-FFAP; 15 m, 0.1 μm film thickness, 0.1 mm inside diameter;Agilent, Pal Alto, CA, USA). Samples were injected in split1:200 mode. The injector and detector ports were set at250 °C. Fatty acid methyl esters were eluted using a temper-ature program set initially at 150 °C and held for 0.25 min,increased at 35 °C/min and held at 170 °C for 3 min, increasedat 9 °C/min, and held at 225 °C for 0.5 min, increased at80 °C/min and finally, held at 245 °C for 2.2 min to completethe run. Total run time was 12.88 min. The carrier gas washydrogen, set to a 30-mL/min constant flow rate.

Chemical Composition—Free Fatty Acid Analysis

Free fatty acids were determined following the method fromAOCS Official Method Ca 5a-40 (2009). Results reported arethe average and standard deviation of triplicates. Approxi-mately 55 g of melted cocoa butter were mixed with hot ethylalcohol (95 %) and the indicator phenolphthalein. The solu-tion was titrated with 0.1 M NaOH. The appearance of a pinkcolor longer than 30 s indicated the neutralization of the freefatty acids. The percentage of free fatty acids is reported on anoleic acid basis.

Powder X-ray Diffraction

The polymorphic form of the cocoa butter was determinedusing a Multiflex powder X-ray diffractometer (Rigaku MSCInc., Toronto, Canada). A pre-heated glass slide (20 °C) with asquare well (0.5 mm depression; side-length, 20 mm) wasfilled with ∼150 μL cocoa butter and analyzed at 20 °C afterforcing the cocoa butter to crystallize at this temperature for

5 min. A copper X-ray tube (λ=1.54 Å, Cu/Kα1) operating at40 kV and 44 mAwas used. A 0.5° diversion slit and scatterslit were used together with a 0.3-mm receiving slit. The X-raydiffraction analysis was carried out covering the region from1° to 35° 2θ at a speed of 1°/min. The powder X-ray diffrac-tion patterns were analyzed with Jade 9.0.1 XRD (RigakuMSC Inc., Toronto, Canada).

Polarized Light Microscopy

The microstructure was determined by polarized light micros-copy using a Leica DM RXA 2 (Leica Microsystems WetzlarGmbH, Ernst-Leitz-Straße, 35578 Wetzlar, Germany). Adroplet of cocoa butter was placed on a pre-heated glass slide(20 °C) and a cover slip was gently pressed down. The slidewas placed in a thermostatically controlled microscope stage(LTS 350, Linkam Scientific Instruments LTD, 8 EpsomDowns Metro Centre, Waterfield, Tadworth, Surrey, KT205HT, England) at 20 °C. Images of the same specimen weretaken after 1 and 10 min on the microscope slide by a Retiga1300i camera set at autoexposure (QImaging, 19535 56thAvenue, Suite 101, Surrey, BC V3S 6K3, Canada). TheOpenlab software 5.5.0 (Improvision, Viscount Centre II,University of Warwick Science Park, Millburn Hill Road,Coventry, CV4 7HS, England) was used to capture the pictureand to adjust the brightness of the pictures. All images wereacquired using a ×10 objective lense (Leica MicrosystemsWetzlar GmbH, Ernst-Leitz-Straße, 35578 Wetzlar, Germa-ny). Micrographs were analyzed using ImageJ (1.44 g,WayneRasband, National Institutes of Health, USA) by measuringtwo parameters: the area and the Feret diameter as reported bythe software. Feret diameters are defined as the distancesbetween parallel tangents touching opposite sides of a 2Dobject. ImageJ reports the longest distance between any twopoints along the region of interest (ROI) boundary. The ROIwas manually defined using the elliptical selection for the areaand the line selection for the diameter from the menu ofImageJ. At least 30 crystals were measured per micrograph.Three micrographs per sample were measured. Averagevalues and standard deviations are reported from all the crys-tals in the three micrographs per sample, but only one micro-graph is shown.

Pulsed Nuclear Magnetic Resonance

The solid fat content was determined by a Bruker minispecmq20 NMR analyzer (Bruker Optics, Milton, Canada) usinglow-resolution pulsed NMR. Approximately 9 mL of cocoabutter were placed inside a 10-mm diameter glass NMR tube.The measurement was performed using one pulse 6 s in lengthfollowing the recommendation of the AOCS Official MethodCd 16b-93 (2000) for nonstable fats.

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Sample Preparation—Tempered Cocoa Butter

After many trials, a protocol was devised to obtain temperedcocoa butter in the βV-polymorphic form containing 4 % solidfat content. The protocol applies to the particular ultrasonicmeasurement cell used in this study (see description below).The procedure starts by melting the cocoa butter in an oven at85 °C for 1 h to remove all crystal memory. Ninety millilitersof material was then cooled statically at room temperature in a100-mL beaker until 60 °C were reached. The ultrasonicmeasurement cell (made of copper) was placed on top of astirring hotplate (Isotemp, Fisher Scientific Company, Ottawa,Canada). A cross-shaped 30-mmmagnetic stirrer at a speed of200 rpm was used ( I so temp, F i sher Sc ien t i f i cCompanyOttawa,, Canada) to gently mix the cocoa butter toprevent it from developing a “hot spot” of temperature. Theultrasonic measurement cell was cooled with water circulatingthrough the cell walls by means of a water bath (RTE-111,Neslab, Neslab Instruments Inc., Newington, NJ, USA). Co-coa butter was poured into the measurement cell when thewall temperature had reached 20 °C. After 150 s, the tem-perature was raised to 25 °C and kept for 27 min and30 s before it was raised to 29 °C. Specimens weretaken for powder X-ray diffraction, NMR and polarizedlight microscopy measurements after 3, 4, and 5 h atthis temperature.

Triplets were prepared and analyzed using 3D plots. Resultsreported are the ones corresponding to one of the three figures.Deviations from these results were estimated to be 5 %.

Sample Preparation—Untempered Cocoa Butter

The protocol for untempered cocoa butter was based onobtaining crystals other than in the βV polymorphic form,with 4 % solids, similar to the tempered sample. The proce-dure starts by melting the cocoa butter in an oven at 85 °C for1 h to remove all crystal memory. 90 mL of material was thencooled statically at room temperature in a 100-mL beaker until60 °C were reached. Cocoa butter was poured into the ultra-sonic measurement cell when the wall temperature reached20 °C. The cocoa butter cooled statically and quickly to 20 °C.After 30, 60, and 90 min samples for the powder X-raydiffraction, the NMR and the polarized light microscopy weretaken.

Triplets were prepared and analyzed using 3D plots. Re-sults reported ones correspond to one figure only. Deviationsto these results were estimated to be 5 %.

Ultrasound Theory

The phase of an ultrasonic wave changes when it travels fromone medium into another one (Dukhin et al. 2005). A simpli-fied explanation of the displacement of a particle as a function

of t, for a particular frequencym, an amplitude xm, and a phaseangle ϕm follows in Eq. 1.

x tð Þ ¼ x⋅cos 2⋅π⋅ f m⋅t þ ϕmð Þ ð1Þ

The term in parentheses is the time-dependent part, alsoknown as the phase of the motion or simply the “phase.” Asimilar equation can be used for the reference oil, with itscorresponding amplitude and phase angle, which will benamed ϕRef,m. To determine the change in the phase angle ofthe sample in relation to the reference, the difference in time,Δtm is investigated using Eq. 2

Δtm ¼ d

υmð2Þ

where d is the distance between transducers, and vm is thevelocity of sound through the material under investigation(cocoa butter) at the mth frequency.

Using Eq. 1, it is possible to compute the phase differencebetween the reference and the samples under study (Eq. 3).

Δϕm ¼ 2⋅π⋅ f m⋅ΔtRef ;m þ ϕRef ;m

� �− 2⋅π⋅ f m⋅Δtm þ ϕmð Þ ð3Þ

By setting Δϕm equal to zero at the center frequency(2.25 MHz in this study), a change of the speed of sound withrespect to the reference material and at a particular frequencycan be determined. In this calculation, the absolute change ofthe phase angle is not considered.

The simplest case is when the phase ϕRef,m and ϕm are set tozero. This case makes the phase angle dependant on the differ-ence in the time of flight for the sample and the reference (Eq. 4)

Δϕm ¼ 2⋅π⋅ f m⋅ ΔtRef ;m−Δtm� � ð4Þ

Equation 4 is relative to the frequency under inspection. Asthe chirp covers a band of frequencies, a figure showing thechanges in the phases as a function of frequencies can begenerated. This allows to present small changes in the speedof sound as a function of frequency.

Ultrasonic Measurements

A SIA-7 (VN Instruments Ltd., Elizabethtown, Ontario, Can-ada) was used to carry out the ultrasonic measurements usingVer. 403102.104800 (2009) of the software.

The copper ultrasonic measurement cell with external di-mensions of 10.0 cm length, 7.5 cm and width, 7.5 cm heightwas cooled by water circulating through the walls.

The receptacle for the sample with dimensions of 4.0 cmlength, 4.0 cm width, and 5.6 cm height was separated fromthe transducers (2.25 MHz/1.0 in.; V-104, Panametrics-NDT,Olympus NDT Canada Inc., Québec City, Québec, Canada)by a polystyrene window 4.0 cm in length. The transducer is

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attached to the polystyrene with an aluminum metal cap andthree screws that provide pressure between the transducer andthe window (Fig. 2).

Lubricant (canola oil) was used in between the transducerand the polystyrene windows to prevent air bubbles, which areknown to strongly attenuate an ultrasonic signal (Saggin andCoupland 2001). The metal caps were designed with twoholes above the transducers to act as a reservoir. As themeasurement cell was heated and cooled, thermal expansioncaused liquid to flow between the transducer and the window.The reservoir helped maintain the liquid layer in order toguarantee no bubbles at the interface.

Each transducer acts as both transmitter and receiver, forwhich a delay time of 30 μs is needed because the transducerscannot execute both functions at the same time. This flexibil-ity allows for more information and data all through themeasurement. Thanks to the dual functionality of each trans-ducer, the ultrasonic signal can be detected in four differentways (Bulman et al. 2012). The data can then be processed toextract different information.

SIA-7 processes the chirp with a bandwidth of 1.7 MHzcenter into a Synthetic Impulse™, which had already beenused to study lipid systems (Martini et al. 2005a). Figure 3

illustrates the Synthetic Impulse™ pattern obtained in trans-mission mode for liquid cocoa butter.

The different times at which the peaks occur indicate if theultrasonic wave transmitted directly through the sample or if itwas reflected at an interface (polystyrene window, sample ortransducer) before it was received by the other transducer. Theposition of each peak in the time domain is given by t=v. d,where v is the speed of sound in m/s, and d is the distancebetween either the interfaces or the transducers, depending onthe surfaces to consider. Using this relation, one can explainthat peak 1 in Fig. 3 represents the transmitted signal directlyfrom one transducer to the other, whereas peak 2 explains areflection at one of the windows.

It is recommended to conduct a quick consistency testbefore every measurement to determine if the transducersare aligned properly and if the lubricant is spread properlybetween the transducer and the window. By calculating thetimes where the peaks should appear for each interface (trans-ducer-window, window-sample) it is possible to check withthe position of the observed peaks if the transducers areproperly coupled to the windows. The SIATool software fromVN instruments operated on a matlab platform was used tocontrol the SIA-7, to acquire the data, to control the water bathand to report the temperature of the three thermometers in themeasurement cell.

The cocoa butter measurements were made in reference tocanola oil, which was used as the liquid control material.

Results and Discussion

Gas–liquid chromatography results showed that palmitic(16:0), stearic (18:0), and oleic (18:1) acid made up themajority of the cocoa butter (Table 1). The measured amountof free fatty acids in the cocoa butter used for this project was0.7 %, almost three times lower than the maximum allow by

Fig. 2 Schematic cross-section of the ultrasonic measurement cell madeof copper with the attached transducers inserted in the metal caps

Fig. 3 Synthetic Impulse™pattern of a transmitted ultrasonicchirp through cocoa butter at 4 %solid-fat content; peak 1 indicatesa transmission signal, and peak 2indicates a reflected signal

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the European Union. In 2000, the European Union had legis-lated that confectionary products should not exceed 1.75 %free fatty acids (European Parliament 2000).

Figure 4 shows the powder X-ray diffraction pattern ob-tained from tempered cocoa butter after 4 h of tempering. Thepeak positions matched the suggested positions for a β-formby Wille and Lutton(1966) and Chapman (1971). Further-more, Le Révérend et al. (2010) stated that the appearanceof a mid intensity peak at a short spacing of 3.75 Å indicates aβV polymorph. Schenk and Peschar (2004) discuss that fourpeaks in the range of 21° to 25° 2θ are indicative for βV

crystals, whereas three peaks indicate a polymorphic form ofβVI. In this case four peaks appear in the named range, whichfortifies the assumption of the presence of the βV polymorph.This leads to the conclusion that the tempering proceduredeveloped here resulted in the βV-form of cocoa butter crys-tals, the desired form for chocolate manufacturers ((Timms1984; Stapley et al. 1999; Padar et al. 2008; Afoakwa et al.2009; Le Révérend et al. 2011; Stortz and Marangoni 2011).

In Fig. 5, powder X-ray diffraction patterns of untemperedcocoa butter are shown. After 30 min in the measurement cell

at 20 °C, cocoa butter was in the α-form (Fig. 5a) after 60 minin the β′-form (Fig. 5b) and after 90 min in the β-form(Fig. 5c). The polymorphic form designation was based onWille and Lutton (1966) and Chapman (1971), who suggestedpeak positions for each of the forms. As the purpose of usinguntempered cocoa butter was to obtain a polymorphic formdifferent from βV, with not too many solids present, we chooseto conduct the ultrasonic measurements during the first 60 minafter pouring the liquid cocoa butter into the measurement cell.

In Fig. 6, polarized light microscopy micrographs of tem-pered cocoa butter collected after 4 h are shown, after 1 min ofcrystallization of the slide (a) and after 10 min of crystalliza-tion (b). In both cases spherulites are observed. The spheru-lites area (A) and Feret’s diameter (d) measured were A=103,00±1,500 μm2, d=115±40 μm after 1 min and A=8,500±4,300 μm2 , d=100±70 μm after 10 min, respectively.

Polarized light micrographs of the untempered cocoa butter(Fig. 7) also revealed spherulites. The sizes of these spheru-lites gave an area A=2,400±1,300 μm2 and a Feret diameterd=36 μm±11 μm after 1 min of crystallization on the slide

Table 1 Fatty acid com-position of cocoa butterused in this study

Fatty acid Area percent (%)

18:0 36.6±0.5

18:1 32.5±0.5

16:0 25.0±0.5

18:2 2.8±0.5

20:0 1.1±0.5

18:1c11 0.4±0.5

16:1c9 0.3±0.5

17:0 0.2±0.5

18:3n3 0.2±0.7

22:0 0.2±0.7

14:0 0.1±0.7

20:c5&8 0.1±0.7

22:1n9 0.1±0.7

22:4n6 0.1±0.7

22:3n3 0.1±0.7

24:0 0.1±0.7

Fig. 4 Powder X-ray diffraction pattern of tempered cocoa butter at 4 %solid fat content and 29 °C

Fig. 5 Powder X-ray diffraction pattern of untempered cocoa buttercrystallized in the ultrasonic measurement cell at 20 °C: 4 % SFC after30 min (a), 8 % SFC after 60 min (b), and 11 % SFC after 90 min (c)

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Fig. 8 Attenuation as a functionof frequency pattern of liquidcocoa butter at 60 °C

Fig. 6 Polarized light microscopy pictures of tempered cocoa butter crystallized on a microscope slide at 20 °C for 1 (a) and 10 min (b)

Fig. 7 Polarized light microscopy pictures of untempered cocoa butter crystallized on a microscope slide at 20 °C for 1 (a) and 10 min (b)

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(Fig. 7a). After 10 min of crystallization, the spherulites grewslightly to showed an area A=3,100±1,900 μm2 and a Feret’sdiameter d=60±50 μm. In addition, the presence of smallspherulites with an area A=67±20 μm2 and a Feret’s diameterd=10±5 μm were observed (Fig. 7b) As untempered cocoabutter has not experienced any external intervention, the driv-ing force to generate crystals was unaltered allowing

nucleation and growing to take place faster than for thetempered material (Marangoni and McGauley 2003). Thus,the larger number of spherulites (~110) present after 10 mi-nutes in the untempered versus the amount in the temperedone (~25) (Figs. 7b vs. 6b). A smoother contour was observedon the cocoa butter spherulites after tempering, whereas theuntempered ones showed rougher edges. The spherulites of

Fig. 10 Attenuation as a function of frequency curves for cocoa butter: a 4 % SFC, untempered; b 4 % SFC, tempered; c 8 % SFC, untempered; and d8 % SFC, tempered

Fig. 9 Attenuation as a function of frequency patterns of untempered (a) and tempered (b) cocoa butter

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tempered cocoa butter showed a well-defined maltese cross inthe middle, which indicates a more ordered spherulite growth,due to the crystallization of selective triacylglycerols. Thismaltese cross could not be seen in the untempered spherulites.It was hypothesized that these differences in the morphology,size and number of spherulites would translate in changes inthe ultrasound measurements.

It is well known that the attenuation of an ultrasonic waveis influenced by the microstructural state of the sample exam-ined. The ultrasound signal is attenuated more by solid fatcompared with a liquid fat and higher frequencies are affectedto a greater extent than lower frequencies (Martini et al.

2005b). Figure 8 shows a 3D attenuation pattern of liquidcocoa butter at 60 °C without crystals in which time, frequen-cy and attenuation are displayed. The x-axis indicates a fixedfrequency range from 0.4 to 3 MHz while the y-axis indicatesmeasurement time that starts at 0 s on the top left corner of theplot. On the right-hand side, the scale for the attenuation isdisplayed (decibels per centimeter). Green means an attenua-tion of 1 dB/cm, yellow of 2–3 dB/cm, and red an attenuationof 5 dB/cm. The horizontal lines in the pictures indicate thesampling times. It can be seen that over a period of 5,500 s nochanges occur in the attenuation for each frequency.

To compare samples with similar amounts of solids, tem-pered and untempered cocoa butter had to be crystallized fordifferent times. Figure 9 shows the attenuation patterns from0 % (top) to 11 % (bottom) solid fat content of untempered (a)and tempered (b) cocoa butter. The scales are different for bothplots but the amount of solids at the end of the run and at thepositions of the inserted arrows in the figure is the same.Differences between the two patterns are evident. The upper-most part of each pattern is not taken into account because theattenuation is caused by the presence of micro-bubbles thatdisappear after certain time after sample insertion (1,000 s for

Fig. 11 Attenuation as a function of frequency patterns at 4 % SFC of untempered (a) and tempered (b) cocoa butter

Fig. 12 Change of phase as a function of frequency for untempered (a) and tempered (b) cocoa butter

Table 2 Attenuation of the ultrasonic signal at a frequency of 1.7 and3 MHz for untempered and tempered cocoa butter at 4 and 8 % SFC

Frequency(MHz)

Untempered(4 % SFC)

Tempered(4 % SFC)

Untempered(8 % SFC)

Tempered(8 % SFC)

1.7 (dB/cm) 3 4.5 4 6

3 (dB/cm) 2 6 3 9

The error in repeatability was estimated at being 5 %

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untempered and 2,000 s for tempered cocoa butter). Below1 MHz, no visible differences can be appreciated betweenboth graphs. Above 1 MHz the attenuation for the temperedsample at the same solid fat content (at 15,000 s) is higher(6 dB/cm) compared with the untempered sample (at 3,500 s,2 dB/cm).

The results obtained from Fig. 9 were further analyzed,comparing horizontal lines drawn at the position that corre-spond to the same solid fat content (see arrows). The attenu-ation vs. frequency results are shown in Fig. 10. Two differentsolid fat contents were chosen: 4 % (Fig. 10a, b) and 8 %(Fig. 10c, d) in accordance to reference values used in the foodindustry (Beckett 2000). All four curves show an increasingattenuation from lower frequencies (0.5 MHz) to about1.7 MHz. At 1.7 MHz, the untempered samples showed adecrease in attenuation for both solid fat contents, while thecurves for the tempered ones continued to increase. Table 2shows the differences between the attenuation values at 1.7and 3 MHz for all the cases displayed in Fig. 10.

In Fig. 11, a section of 50 s from Fig. 9 of untempered (a)and tempered (b) cocoa butter is shown, where both sampleshave a SFC of 4 % to compare the results more easily.

At frequencies higher than 1.5 MHz, the tempered sampleshows a greater attenuation compared with the untemperedone. At 2.5 MHz, the untempered sample shows 2 dB/cmattenuation whereas the tempered cocoa butter attenuates thesignal by 5 dB/cm.

In Fig. 12, the change in the phase of the ultrasonic wave(Eq. 4) at different frequencies is shown for untempered andtempered cocoa butter. The phase of untempered cocoa butter(Fig. 12) changes less than 0.5 rad over the whole frequencyand time range. For the tempered cocoa butter (Fig. 12), thechange in phase is more pronounced as the solid fat contentincreases (bottom of plot in Fig. 12b, 11 % at 18,000 s).Furthermore, a change in the phase of more than 1 rad canbe seen at 3 MHz (right bottom corner of Fig. 12b). In bothpictures the black line indicates a solid fat content of 4 %. Nophase changes are observed at high frequencies for the untem-pered sample (Fig. 12a). The tempered cocoa butter (Fig. 12b)showed a phase change of 0.5 rad at a frequency of 3 MHz. Itis hypothesized, that the changes in phase as a function offrequency occur due to changes in polymorphism but moreexperiments are necessary to corroborate this statement.

Conclusions

We have shown that the velocity, attenuation, and phase of anultrasonic chirp wave can be used to detect differences be-tween tempered and untempered cocoa butter. Here, we de-veloped a protocol to produce tempered and untemperedcocoa butter in a reproducible fashion and determined thatultrasonic wave attenuation and phase angle changes as a

function of frequency could be used to differentiate betweentempered and untempered cocoa butter using canola oil as areference. The results show that the ultrasound signal is atten-uated more in a solid state than in a liquid one and that at3 MHz the signal is more attenuated than at 1.7 MHz. Atfrequencies higher than 1.5 MHz the attenuation for the tem-pered sample at 4 % SFC is higher, 6 dB/cm, compared withthe untempered sample, 2 dB/cm.We also demonstrated that apositive phase angle change appears for tempered cocoa butterwhile a negative one appears for untempered cocoa butter.Based on these results, we propose that measurements of thefrequency dependence of the chirp wave phase angle at fre-quencies higher than 2 MHz could be used to monitor theproper tempering state of cocoa butter and possibly chocolate.

Acknowledgments This work was supported by the Natural Sciencesand Engineering Research Council of Canada (NSERC) and by Herzog-Carl-Scholarship from the University of Hohenheim, Germany.

The authors would like to thank Dr. Lyn Hiller of the Department ofHuman Health and Nutritional Sciences at the University of Guelph forher help with the GC analysis.

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