THE RELATIONSHIP B E T W E N POLYMORPHISM, CRYSTALLIZATION

KINETICS, AND MICROSTRUCTURE OF STATICALLY CRYSTALLIZED

COCOA BUTTER

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

SARA ELIZABETH McGAULEY

In partial fulfiilment of requirements

For the degree of

Master of Science

August, 2001

O S.E. McGauley, 200 1

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ABSTRACT

THE RELATIONSHIP BETWEEN POLYMORPHISM, CRYSTALLIZATION KINETICS, AND MICROSTRUC'TZTRE OF STATICALLY CRYSTALLIZED COCOA

BUTTER

Sara E. McGauley University of Guelph, 2000

Advisor: Professor Alejandro G. Marangoni

Differential scanning calorirnetry @SC) and powder X-ray diffraction (XRD) were used

to detemiine the polymorphic form of cocoa butter crystallized staticaily. From these

results, a time-temperature state diagrarn was consû-ucted. Isothennal crystallization

curves were obtained by measuring changes in solid fat content (SFC) as a function of

tirne. Crystallization kinetics were quantified using the Awami model. A significant

difference was observed in the Avramî exponent (n) above and below 20°C. Polarized

light microscopy (PLM) was used to image the microstnicture of the various polymorphic

forms of cocoa butter in different regions of the state diagrarn. Various microstructures

could usually be associated with one polymorphic form as a result of different processing

conditions. The microstructure was quantified from PLM images using the particle-

counting mass fiactal dimension (D). Microstructures below 20°C had a fractal

dimension of 2.12, while at 20°C and above a fiactal dimension of 2.30 was obtained. A

strong correlation was found between n and D (8 = 0.95).

ACKNOWLEDGEMENTS

1 would like to take this opportunity to thank my advisor Dr. Alejandro

Marangoni for his guidance, support and patience. 1 would also like to thank my

cornmittee members (Drs. H. D. Goff and Y. Kakuda). A special thanks to Dr. H. D.

Goff for letting me use his DSC for several rnonths.

I thank my fellow lab mates Arnanda Wright, Rob Blenkinsop, Leslie Copp,

Nadia BrunelIo, Rodrigo Campos, Suresh Narine, Geoff Rye, Anand Singh, Jolm Craven,

Jenold Litwinenko and Gianfianco Mazzanti for their help and fkiendship. 1 am

especially grateful to Amanda Wright for always listening and giving good advice.

1 would Iike to acknowledge Ken Baker for his guidance with the microscopy part

of this project. As well, 1 would like to thank Dr. M. Jennings for his help with the X-ray

diffraction expenments.

To my mom, dad and sister, thank you for your love and support,

especially during the last few rnonths. Finally, I wouid like to thank Greg for his help

with this thesis and for his support, friendship and encouragement during the ups and

downs of the last few years.

T-LE OF CONTENTS

A C m O WLEDGEMENTS ................................................................................................. i

. . TABLE OF CONTENTS ........................ .... ...................................................................... il

LIST OF TABLES ........................................................................................................... iv

LIST OF FIGURES ............................................................................................................ v

INTRODUCTION ..............-..-. ... ................................................................................... 1

LITEIRATURE REVEW ................................................................................................... 5

Introduction .................................................................................................................... 5

Triacylglycerols .............................................................................................................. 5

Polymorphisrn ............................................................................................................... IO

Cocoa Butter Polymorphism ......................................................................................... 22

Crystallization of Fats ................................................................................................... 29

Microstructure ............................................................................................................... 3 4

Fractals .......................................................................................................................... 39

OBJECTNES ...................... .... .................................................................................... 46

MATERIALS AND METHODS ...................................................................................... 48

......................................................................................................... Source Materials 48

Triacylglycerol Profile ................................................................................................. 48

Fatty Acid Profile ............... .. ..................................................................................... 45

Fatty Acid Content ........................................................................................................ 49

Phosphorous Content .................................................................................................... 49

Solid Fat Content Determination .................................................................................. 50

Differential Scanning Calorirnetry .................. .. ..................................................... 50

............................... Powder X-Ray Diffiaction ... ...................................................... 51

.......................................................................................... Polarized Light Microscopy 52

Image Processing and Particle Counting ...................................................................... 53

Statistical Analysis ........................................................................................................ 53

....................................................................................... RESULTS AND DISCUSSION 55

. . Chernical Composition .............. .. ............................................................................ 55

............................................................................ Polymorphism ............................ ... 56

.................................................................... ................... Crystallization Kinetics .... 71

............................................................................................................... Microstructure 81

. . ......................... Other Processing Conditions ..... ......................................................... 102

............................................................................................................. CONCLUSIONS 107

.............................................................. SUGGESTIONS FOR FUTURE RESEARCH 109

............................................................................................................... REFERENCES I l l

LIST OF TABLES

Table 1: Polymorphic forms of POP (Sato et al., 1989; Yano et al., 1993, POS

(Arishima et al., 1991) and SOS (Sato et al., 1989; Yano et al., 1993) and their

melting temperatures (Tm) .................................. ,... . . . ............................. 18

Table 2: Polymorphic forrns of cocoa butter as determined by various research groups 26

Table 3: Avrami exponent values for the different types of growth and nucleation

(Sharples, 1966) .............................................................. . ................................ 3 4

Table 4: Fatty Acid Composition of Cocoa Butter ........................................................ 55

Table 5: Triacylglycerol Composition of Cocoa Butter .......... ........................................ 55

Table 6: Free Fatty Acid and Phospholipid Content of Cocoa Butter. ........................... 56

Table 7: Characteristic short spacings as determined by XRD for the various

polymorphic foms of cocoa butter (Larsson, 1994). ............................................... 63

Table 8: Solid Fat Content (SFC) for "cold tempered" and directly crystallized cocoa

butter held at SOOC, 24OC and 26°C for 28 days. ............................................... 102

Table 9: The relationship between the n and r, D and s, as well as D and n for statically

crystallized cocoa butter. ......... . . . ....... ...... ... .... . . ........ . . . . . . . . . . . . . .. . . 107

LIST OF FIGURES

....................................... Figure 1: Schematic of idealized fat crystal network.. .2

Figure 2: The influence of the various levels of structure in a fat network on the

macroscopic rheological properties.. ................................................... -3

.................................. Figure 3: General structure of a triacylglycerol rnolecule.. -6

Figure 4: The hydrocarbon subcell packing of the B, P' and a

........................................................................ polyrnorphic forms.. 13

......................................... Figure 5: Double- and triple-chain-length structures.. 15

Figure 6: Structural models of polymorphic transformations

............................................................................ in POP and SOS 20

Figure 7: Particle counting involves the determination of the number of reflections for

...................................................................... various box lengths ...YI

Figure 8: Overlay of three characteristic melting profiles obtained by DSC of the

.......................................... different polymorphic forrns of cocoa butter ..58

Figure 9: Peak melting temperature as a function of crystallization temperature

obtained fiom DSC melting profiles of cocoa butter statically crystallized

................................................................................. for 7 days.. ..6 1

Figure 10: Characteristic X-ray diffraction patterns of the various polyrnorphic

forms of cocoa butter crystallized at -20 OC for 2 minutes,

............................. 5 OC for 2 minutes, 5 OC for 5 days and 22 OC for 28 days 64

Figure 11: Tirne-temperature state diagram for the polymorphism of statically

................................................................ crystallized cocoa butter.. .66

Figure 12: XRD pattern of cocoa butter statically crystallized at 26°C for 1 day ........ -69

Figure 13: Crystallization curves of cocoa butter crystallized at -20°C,

............................................. 10°C, 15°C and 17.5"C, 20°C and 22S0C.. 73

Figure 14: Changes in the Avrami exponent as a fimction of

.............................................................. crystallization temperature. -75

Figure 15: Induction times for statically crystallized cocoa butter as a

................................................. function of crystallization temperature.. 78

Figure 16: Cornparison of the polymorphic forms as determined fiom

peak temperatures obtained h m DSC melting profiles and

................ fiom crystallization curves of statically crystallized cocoa butter.. ..80

Figure 17: Images obtained by PLM of the alpha form of cocoa butter

crystallized at -20 OC for 1 day, -20 O C for 7 days,

-15 OC for7 days, andO°C for 1 day ................................................... 82

Figure 18: Micrographs of the P' form obtained by static crystallization

at 0°C for 14 days, 10°C for 5 days, 15OC for 14 days,

............................... 20°C for 1 day, 22OC for 1 day, and 24°C for 3 days.. ..83

Figure 19: Images of the stable B form of cocoa butter statically

crystallized at 20°C for 28 days, 22°C for 28 days,

...................................................................... and 26OC for 28 days -85

Figure 20: PLM images of cocoa butter statically crystallized at 0°C

for 1 day, 5 days, 7 days, 14 days, 21 days,

................................................................................. and 28 days -89

Figure 21: PLM images of cocoa butter crystallized at 15°C for

......................................... 1 day, 7 days, 14 days, 2 1 days, and 28 days.. .9 1

Figure 22: Polarized light microscope images of cocoa butter statically

............. crystallized at 20°C for 1 day, 5 days, 7 days, 2 1 days and 35 days.. -93

Figure 23: Images obtained by PLM of cocoa butter crystallized at

..................................... 26°C for 1 day, 3 days, 7 days, 14 days, 28 days. 95

Figure 24: Fractal dimension vs. crystallization temperatures of

....................................... cocoa butter statically crystallized for 7 days.. -98

Figure 25: Fractal dimension as a function of time of cocoa butter

.............................. statically crystallized at-20°C, 5"C, 20°C, and 26°C.. 100

Figure 26: The fiactal dimension determined microscopically

........................................................ vs. crystallization temperature. .10 1

Figure 27: Polarized light microscopy images of cocoa butter crystallized

at -15°C for 2 days and then held at 20°C and 24OC

for 28 days and of cocoa butter crystallized at 5OC for 2 days

....................................... and then heId at 20°C and 24°C for 28 days.. -104

vii

INTRODUCTION

Both chemical and physical properties of cocoa butter have been shown to

influence the quality and acceptability of confectionary products. Processing conditions

affect the crystallization of the cocoa butter triacylglycerols (TAGs) into a particular

polyrnorphic state, which in turn influences the microstnicture and macroscopic

properties of the network. Fat crystal networks are comprised of branched and B

interlinked particles which form a three dimensional network, the voids of which are

filled by liquid fat. The particles, which represent aggregates of crystallites, aggregate to

form clusters. The clusters pack in a regular homogenous manner and represent the

largest structural building block of the fat crystal network (Figure 1). The macroscopic

properties of the network determine final athibutes such as viscosity, dernolding, snap,

surface gloss and desired rnelting characteristics of the confectionary product. The

levels of structure found in cocoa butter and their influence on macroscopic rheo!ogical

properties are shown in Figure 2.

Many researchers have studied the effect of lipid composition and polymorphism

on macroscopic rheological properties but have not suficiently considered the

importance of the microstructure of the network. Depending on the source and the

refining process, the chemical composition of cocoa butter rnay vary (Dimick, 1999).

This has been shown to influence crystallization rates and hardness characteristics

(Chaseri and Dirnick, 1989). As well, the TAG composition of cocoa butter directly .

influences the polyrnorphism of the network. The type of polymorphism dictates the

melting range of the fat network.

Crystallite

\

Figure 1: Schematic of idealized fat crystal network.

Figure 2: The influence of the various levels of structure in a fat network on the macroscopic rheological properties.

Macroscopic Properties

Microstructure

Polymorphism Processing Conditions

A

I Molecular Structure

Within the microstructurd Ievel, the polymorphic forrn of cocoa butter has also been

s h o w to influence the shape and size of the crystals (Vaeck, 1960). The structure of the

solid state is particularly important for cocoa butter. Six different poiymorphic forms are

generally recognized for cocoa butter each with its own characteristic melting point,

stability and morphology.

Even though the effects of Iipid composition and polyrnorphism on final product

quality have been extensively studied, they alone cannot be used to predict the

macroscopic properties of a fat. UntiI recently, cocoa butter microstructure had only

been studied qualitatively. The microstructure of the unstable polymorphic form of

cocoa butter has been described as a bright crystdline mass (Vaeck, 1960) whereas the

more stable forms have been associated with various microstructures as described by

Manning and Dimick (1985). As well, no comprehensive study has been carried out to

determine the effect of polymorphic transformations on microstructure as a function of

both tirne and temperature under static conditions. The importance of the microstructural

level of the network in the determination of macroscopic properties has led to the

development of a theory that allows for the quantification of this level of structure

(Marangoni and Rousseau, 1996; Narine and Marangoni, 1999a). Analysis of the

microstructure using fracta1 geometry has also been related to mechanicd properties of

the network and therefore provides information about certain sensory attributes.

Understanding al1 of these levels of structure (Figure 2) and how they are influenced by

processing conditions will hopefully aid in the prediction of the macroscopic properties

of confectionary products.

LITERATURE REVlEW

Introduction

A review of the scientific research relating to the various Ievels of structure of a

fat network will be presented in this chapter. Triacylgfycerois are h o w n to crystallize in

a number of different ways depending on extemal factors and composition as a resuit of

polyrnorphism. Cocoa butter is a fairly complex system in that six different polymorphs

have been observed. However, the distinct nature of these polymorphic foms is still an

area of conboversy in confectionary research. In a few cases, distinct microstructures

have been associated with a particular polymorphic fonn. This section reviews the

effects of triacylglycerol composition on physical properties, polymorphic forrns and

their resultant morphologies, as well as a model to quanti@ the microstructure of a fat

crystal network.

Triacylglycerols

The major constituents of fats, including cocoa butter, are triacylglycerols

(TAGs). TAGs are com.posed of a variety of fatty acids esterified to a glycerol backbone

(Figure 3). There is a wide range of triacylglycerols found in nature due to the positional

isomerism of different fatty acids within the glyceride molecule. The fatty acid chains

may be saturated, unsaturated, branched or iinear, short or long and contain either odd or

even carbon numbered fatty acids (Srnall, 1986). Shorter chains are found in ruminant

milkfat whereas longer chains are found in vegetable oils. Saturated chains tend to adopt

a straight conformation with the carbon atorns lying zig-zag in a plane. In general,

unsaturated fatty acids found in vegetable oils contain one or more cis double bonds.

Figure 3: General structure of a triacylglycerol molecule.

In this case, the bend that occurs at the double bond determines which fatty acids pack

next to one another (Blaurock, 1999). TAGs with identical fatty acid chains are termed

mono-acid TAGs, while those with more than one type of fatty acid are called mixed-acid

TAGs (Timnis, 1984).

The smallest level of charactenstic structure present in fat crystd networks is

found at the molecular level, as shovm in Figtue 2. The determination of both

triacylgIycerols and fatty acids has been established for fat systems (Christie, 1982).

Triacylglycerols have been well studied and the bond angles and bond lengths of the

atoms within each rnolecule have been established based on structural organic chemisq.

However, the method by which a group of TAGs forms a particular crystal structure is

unknown. The complexity and flexibility of the TAG molecules allows for different

crystdline packing of the same group of molecules resulting in polymorphism.

Molecular modeling has been used to try and explain the packing of triacylglycerols into

particular polymorphic forms (De Jong and van Soest, 1978; De Jong et al., 1991;

Hagernann and Rothfus, 1983; Hagemann and Rothfis, 1992; van Soest et al., 1990; Yan

et al., 1994). However, very little work has been carried out in relating TAG composition

to macroscopic properties. TAG composition is known to have an effect on the resultant

polymorphisrn, which in tum affects melting characteristics. Narine and Marangoni

(1 999b) and Marangoni (2000) have created a mode1 that indirectly relates lipid

composition to macroscopic properties. However, no predictive method exists that

relates triacylglycerol composition to melting profiles.

The rnolecular composition of a fat influences various physical characteristics

including temperatures of crystallization, melting point, and polymorphic behaviour.

Cocoa butter is mainly composed of symmetric triacylglycerols with oleic acid at the sn-2

position including POP, SOS and POS where P stands for palmitic acid, S for stearic acid

and O is oleic acid. It contains approximately 20% triacylglycerols that are liquid at

room temperature and has a mehing range of 32-35°C (SchIichter-Aronhime and Garti,

1988). However, this melting range is dependent on polyrnorphism with the least stable

form melting 2t a lower temperature. Cocoa butter contains oniy trace amounts of

unsyrnmetrical triacylglycerols such as PPO, PSO and SSO (Shukla et al., 1983). The

predomuiant fatty acids that have been found in cocoa butter are oleic, palmitic and

stearic acids, while linoleic, arachidic, and myristic acids constitute a very small

proportion (Chaiseri and Dimick. 1989; Shukla et al., 1983; van Malssen et al., 1996b).

Methods have been established in order to determine the TAGs present in cocoa butter.

EarIy studies used fiactional crystallization (as reported by Hilditch and Williams, 1964),

counter current distribution (Dutton et ai., 1961 ; Scholfield and Dutton, 1959; Scholfield

and Hicks, 1957), and various oxidation techniques (Kartha, 1953; Pnvett and Blank,

1963; Youngs, 196 1). As well, determination of the fatty acid composition after lipase

hydrolysis dlows for the determination of TAG composition (Coleman, 196 1). However,

these compositional anaiysis techniques have been replaced by more modern

chrornatographic techniques. Gas liquid chromatography (GLC) has been successfùlly

used to detect the TAG present in fats (Padley and Timms, 1980; Young, 1 984). This

technique provides information on the composition of a fat according to the carbon

nurnber of the triacylglycerols. The composition of cocoa butter has also been

determined by high performance liquid chromatography (HPLC) (Manning and Dimick,

1 985; Shukla et al., 1983 ; Shukla, 1 995). This technique allows for the identification of

each individual TAG instead of simply providing the carbon number, which could

correspond to several different TAGs.

Cocoa butters fiom different sources have been shown to have different chernical

compositions and hence different physical properties (Chaiseri and Dimick, 1989; Shukla

et al., 1983). The largest difference between cocoa butters is found in the content of

mono-unsaturated (POP + POS + SOS) and di-unsaturated (POO + SOO) triacylglycerols

(Shukla et al., 1983). Cocoa butter samples with high concentrations of di-unsaturated

TAGs have been shown to be soft, as a result of the double bonds in the fatty acids in the

sn-3 position causing extra kinking in the structures that intempt molecular packing of

the major components (Chaiseri and Dimick, 1989). The triacylglycerol composition of

three different cocoa butters fiom Malaysia, Brazil and Ghania was determined. The

Mdaysian cocoa butter was found to contain high amounts of mono-unsaturated

triacylglycerols and low amounts of other unsaturated triacylglycerols while the Brazilian

cocoa butter contained low arnounts of mono-unsaturated triacylglycerols and high

amounts of other unsaturated triacylglycerols (Shukla et al., 1983). This compositional

variation resulted in differences in solid fat content, crystallization behaviour and melting

points (Shukla, 1995).

Most fats used in industry are refined before they are used as food ingredients.

However, cocoa butter is obtained by hydraulic pressing of the cocoa bean cotyledons

resulting in an unrefined product. Pure pressed cocoa butter contains many lipid species

other than the triacylglycerol component and these minor components, including

diglycendes, monoglycerides, fiee fatty acids and sterols, have been shown to affect

crystallization behaviour and final product quality (Dimick, 1999). For some products

such as white chocolate, the flavour associated with unrefined cocoa butter is regarded as

unpleasant and in th is case refined cocoa butter is used (Beckett, 2000). The refining

process may include bleaching, degumming, neutraiization, deodorization, or

fractionation. These treatments generally reduce the fiee fatty acid content and remsve

more polar components, including glycolipids and phosphatides (Dimick, 1999). It has

been determined that polar molecules such as phospholipids crystallize preferentially.

The polarity of these lipids may make it energetically favourable for them to crystallize

fkom the nonpolar melt. A crystallization mechanism has been proposed that suggests

that polar lipids may play a significant role in crystal seed nuclei formation events. The

amphiphilic nature of these polar lipids may be the basis of their association in early

crystallization events. A study by Lawler, (as reported by Dimick, 1999), on the

crystallization behaviour of degurnrned cocoa butter followed by the addition of

phospholipid material supports this mechanism. Degurnmed cocoa butter was found to

have significantly lower crystallization rates compared to unrefined cocoa butter. When

the phospholipid material was added back to the refined cocoa butter, the nucleation

induction was reduced and the crystallization growth rate was increased (as reported by

Dimick, 1999). This suggests that refined fat systerns will crystallize in a different

manner than unrefined ones.

Polymorphism

Polymorphism, defined as the existence of two or more crystalline stmctures for

the sarne substance, has been well established in pure triacylglycerols and natural fats.

Different polyrnorphs have very different physical properties but, upon melting, give

identical liquids. The existence of different crystalline structures in triacylglycerols was

known as early as 1849, with the discovery that tristearin had two distinct melting

temperatures (as reported by Chapman, 1962). A few years later, tristearin was shown to

have three melting points (Duffy, 1853). The nurnber of melting points reported for

tristearin oscillated between two and three over the next severai decades. This was

believed to be the result of isomerism (Hagemann, 1988). Controversy over the number

of melting points of tristearin was most likely the result of poorly characterized

triacylgIyceroIs that were of a low purity due to a lack of modem chromatographie

methods.

Four different solid forms of tristearin were identified in the 1930s using X-ray

difiaction (XRD) (Clarkson and Malkin, i934). It was established that the ba i s for the

multiple melting temperatures observed for a single substance was the result of

polymorphism (Clarkson and Malkin, 1 93 4). However, later research contradicted the

associations made by Clarkson and Malkin (1934) between the melting points and the

XRD patterns and demonstrated that only three forms of tristearin were possible (Filer,

1946; Luaon, 1946). The three distinct XRD patterns for tnstearin were confirmed using

infkared spectroscopy (Chapman, 1955). The controversy between these two sets of

findings was M e r complicated by the fact that the same syrnbols were used to

designate different polymorphic forms and the fact that no crystal similarities or

differences were described for any of the polyrnorphic forms. Presently, the polymorphs

determined by Lutton (1 946) and the XRD patterns obtained by Clarkson and Malkin

(1 934) are used to identify polymorphic forms.

Polymorphism arises fiom the possibility of variations in hydrocarbon chah

packing. The mode of packing of the hydrocarbon chains can be described using the

subcell concept. #en a fat crystallizes, triacylglycerol molecules adopt a specific

conformation and packing arrangement in order to optimize intra- and intermolecular

interactions and achieve an efficient packing (Larsson, 1994). The smallest building unit

of a crystal is referred to as the unit ce11 and the repetition of this unit in its three axial

directions gives rise to a crystal lattice. The subcell on the other hand, is a smaller

repeating unit within the unit cell. The unit ceIl is relatively large in long chah

hydrocarbons but the subcell geometry c m be determined using powder XRD (Larsson,

1994; Latvler and Dimick, 1998; Timms, 1984). Only seven different subcells are

necessary to include al1 of the possible crystal structures.

The method of choice for the study of fat polymorphism is powder XRD but

other rnethods such as differential thermal analysis @TA), differential scanning

calorirnetry (DSC), infrared spectroscopy (IR) and microscopy may also yield useful

information on crystal structure (deMan, 1992). When fats are analyzed by powder

XRD, two types of spacings are observed, namely long spacings and short spacings.

Long spacings correspond to reflections originating fiom planes formed by the methyl

end groups of the triacylglycerols and are dependent on the chain length and the angle of

tilt of the component fatty acids. Short spacings, on the other hand, are sensitive to the

cross-sectional packing of the hydrocarbon chains and are independent of chain lenath

(deMan, 1 992).

It is well accepted that fats contain solid TAGs in three major polymorphic fomis.

The f o m with the highest melting point and the greatest stability is called the P

polymorph. This has a tnclinic subcell (Figure 4A) with parallel hydrocarbon-chain

planes and has a short spacing at 4.6A.

Hexagonai

Figure 4: The hydrocarbon subcell packing of die B (A), PY(B) and a ( C ) polymorphic forms.

The p' polymorph displays intermediate stability and has a packing structure, which is

orthorhombic with perpendicular chah phases and short spacings at 3.8A and 4.2A

(Figure 48). Finally, the hexagonal subcell denoted as the a polymorph is the least stable

and has a short spacing at 4.1 SA (Figure 4C). The subcell of the a polymorphic form

appears as a close packing of oscillating fatty acid chains (Hagemann, 1988; Larsson,

1994; Lawler and Dimick, 1998; Sato, 1996; Timms, 1984). In mixed triacylglycerol

systems, other metastable polyrnorphs called the y, and 6, and several B' polyrnorphs

have been observed (Sato, 1996). The y polymorph, like the P' form, is orthorhombic

perpendicular with characteristic short spacings at 3.8A and 4.2A (Larsson, 1994).

TAGs may also display variations in the tilt of the hydrocarbon molecules. This is

a phenornenon known as polytypism. This results in the existence of more than one form

of each polymorph and means that within groups having the same subcell, different

melting points can be obtained. Differences in polytypism are noted by a progressively

higher subscript with decreasing melting point following the polyrnorphic designation

(Hagemann, 1988; Larsson, 1994; Lawler and Dimick, 1998). Long chain TAGs are

thought to pack side by side in separate layers. It has been suggested that the TAG

molecule forms a chair-shape structure and, upon crystallization, the TAGs arrange in

pairs, head to tail. Two packing modes are possible. The double-chain-length or bilayer

structure is formed when the chemical structure of the fatty acids is very similar (Figure

SA).

Figure 5: Double- (A) and tripie-chain-length (B) structures.

The triple-chah-length or trilayer structure occurs when one of the fatty acids is largely

different h m the others or of a different chah length (Figure 5B). The trilayer structure

is thought to be the result of chain sorting (Larsson, 1994; Lawler and Dimick, 1998;

Sato, 1996). During polymorphic transformations a bilayer structure may convert to a

trilayer structure and in some cases a trilayer may convert to a bilayer (Larsson, 1994;

Sato, 1996). Bilayer and trilayer structures of the TAG are indicated with a -2 and -3,

respectively, following the polyrnorph designation (Hagemann, 1988; Lawier and

Dimick, 1998; Timms, 1984).

Physical properties of TAGs, such as polymorphism, melting and transition

temperatures and viscosity, are generally known but specific and detailed crystal

structures are largely lacking. Only the structure of even chain length mono-acid P

polyrnorph fiorn single crystal x-ray studies have been confirmed. The triacylglycerols

used in this determination were tricaprin (Jensen and Mabis, 1963; Jensen and Mabis,

1 966) and tnlaurin (Larsson, 1964; Vand and Bell, 195 1). The structure of a single

complex TAG, tri- 1 1 -brornoundecanoin has also been determined by single-crystal XRD.

Its structure is similar to that of trilaurin but with different packing within the crystal

lattice because of the uneven fatty acid chah lengths (Larsson, 1963). More recently, the

structure of the p form of the mixed-acid TAG 1,2-dipalmitoyl-3-acetyl-sn-glycerol was

determined to be markedly different fkom that of mono-acid triacylglycerols. This was

the first analysis of the structure of a TAG of triple chain length (Goto et al., 1992). The

structure of the p' has yet to be determined. It is known to form thin needles with a

tendency for twinned growth, which makes the detemination of single-crystal structure

complicated. The main features of this form have been determined by Minned crystal x-

ray analysis of triundecanoin (Hernqvist and Larsson, 1982) and LML (L: lauric acid, M:

myristic acid) (Birker et al., 1991). The molecular structure and packing arrangement of

the a polyrnorph remain fairly elusive. It has only been determined that the subcell

stnicture is hexagonal and that the molecules are organized in a double-chain length

structure (Hagemann, 1 98 8; Larsson, 1994; Sato, 1 996).

Little is known about the crystal structure of rnixed-acid triacylglycerols and their

behaviour is ofien explained using rcsults from studies involving saturated monoacid

triacylglycerols. However, the investigation of mixed-acid triacylglycerols has occupied

many researchers (Arishima et al., 1991 ; Boubekri, 1999; Engstrom, 1992; Fahey et al.,

1985; Filer et al., 1946; Gibon et al., 1986; Goto et al., 1992; Jackson and Lutton, 1950;

Kodali et al., 1987; Koyano et al., 1989; Koyano et al., 1991 ; Landman et al., 1960,

Larsson, 1972; Lovegren et al., 197 1 ; Minato et al., 1997; Sato et al., 1989; Takeuchi et

ai., 2000). These researchers have determined the polymorphism and polymorphic

transformation of various mixed-acid TAGs. To fully understand the polymorphism of

cocoa butter, it is necessary to understand the crystallization behaviour of individual

TAG molecular species. In particular, determination of the polymorphic modifications of

1,3-di-palmitoyl-2-oleoyl glycerol (POP), 2-oleoyl-palmitoyl-stearoylglycerol (POS) and

1,3-di-steroyl-2-oleoyl glycerol (SOS) is necessary as these are the main triacylglycerols

found in cocoa butter. In early studies, three polyrnorphic forrns of POP (Daubert and

Clarke, 1944; Filer et al., 1946; Landmann et al., 1960; Lavery, 195 8; L-Won, 1946;

Jackson and Lutton, 1950) and three forms of SOS (Lavery, 1958; Lutton, 1946) were

thought to exist. Four forms of POP and SOS were also determined (Malkin and Wilson,

1 949). However, in recent studies, six polymorphic forms of POP (Gibon et al., 1 986;

Sato et al., 1989; Yano et al., 1993) and five forms of SOS (Sato et al., 1989; Yano,

1993) have been determined, as shown in Table 1. Three forms of POS were originally

determined (Lutton, 195 1) but it is now accepted that four polyrnorphic forms exist

(Arishima et al., 1991 ; Lavery, 1958, Landrnann et al., 196O), as s h o w in Table 1.

Table 1: Polymorphic foms of POP (Sato et al., 1989; Yano et al., 1993), POS (Anshima et al., 1991) and SOS (Sato et al., 1989; Yano et al., 1993) and their melting temperatures (Tm)

~ o l p o r p h i c OC Y s P2' Pr' Pz P i

form of POP Tm(OC) 15.2 27.0 29.2 30.3 33 -5 35.1 36.7

Polymorphic a Y P' P z Pr form of SOS

Trn(OC) 23.5 3 5 -4 36.5 41 .O 43 -0

Polymorphic a 5 P' P f o m of POS

Trn("C) 19.5 28.3 31 -6 35.5

Studies using 99.9% pure TAGs, to elirninate any polyrnorphic behaviour that

results from impurities, have been carried out. Using XRD, DSC and Raman

spectroscopy, remarkable differences were found between monosaturated acid TAGs and

mixed-acid TAGs in terms of chain length structure, subcell structure, methyl end

packing and glycerol group conformation. It was determined that steric hindrance

between the saturated and unsaturated acyl chains leads to complexity in the lateral

packing of the larnellar-type crystal structure. However, no single crystal analysis has

been successful, so precise structures on the molecular level are still unknown (Arishima

et al., 199 1 ;Sato et al., 1989). Fourier transform infiared spectroscopy was used to study

polymorphic transformations of POP, SOS and POS in order to determine the molecular

stnicture of their subcelis, olefinic conformations, methyl end packings and the glycerol

groups (Yano et al., 1993). This work c ~ ~ r r n e d the main molecular structure of the five

polymorphs of SOS (Table 1). A double-chain-length structure was observed in the a

form of al1 three of these mixed-acid TAGs and the two P' forms of POP. Al1 of the

other polymorphic forrns of POP, POS and SOS were found to have a triple chah length

structure (Yano et al., 1993). Figure 6 illustrates the polymorphic transfomation that

occur in POP and SOS. Usually, once a transformation fiom a double- to a triple-chah

length structure has occurred it stays in this form as shown for SOS. In POP, a transition

occurs fkom a double chain length structure (a) to a triple (y) and back to a double chain

length (P') structure again. Although the mechanism of this transformation is unclear,

the palrnitoyl and oleoyl chahs of the double chain-length structure are thought to

accommodate the sarne lamellae giving rïse to stenc hindrance, resulting in a deformed

subcell in the P' form of POP (Yano et al., 1993).

a Double

POP)

Triple P ' Double

Figure 6: Structural models of polymorphic transformations in POP and SOS (Yano et al., 1993).

Binary and ternary systems of triacylglycerols have been found to demonstrate

cornplex behaviour. Research done in this area before 1965 was reviewed by Rossel

(1 967) and classified in terms of mixtures of monoacid triacylglycerols and mixtures of

rnixed-acid triacylglycerols. Many of these results may not represent true equilibrium

States due to the questionable purity of the samples and isomer contamination. Therefore,

caution must be exercised in the interpretation of these results. Saturated simple

triacylglycerols, mixtures of unsaturated and saturated monoacid triacyIglycerols,

systems of a simple triacylglycerol and one mixed-chah triacylglycerol and systems of

mixed chain triacyIglycerols have been investigated (as reported by Hagemann, 1988).

The study of binary mixtures, including POP, POS, and SOS is of particular interest in

confectionary products. WiIle and Lutton (1 966) examined a binary system of POS/SOS

and at 75% POS the polyrnorphism was reasonably close to that of cocoa butter. A solid

solution was observed for al1 three polymorphic foms (a, P', and B) but there is some

controversy about the existence of a eutectic for the p phase (Ollivon and Perron, 1992).

Also, binary mixtures of POP/ POS, and POP/ SOS have been shown to form a eutectic

with incomplete miscibility. although al1 three TAGs are completely miscible in the solid

state in the proportions present in cocoa butter (Timms, 1984). This was confirmed in a

study of a ternary system of POP/POS/SOS at the same relative ratio as that in cocoa

butter. This system was found to crystallize in a perfect mixed crystal and exhibited two

p foms, which were identical to those found in POP and SOS (Sato, 1996). Recently,

DSC and polarïzed light microscopy (PLM) were used to constmct phase diagrams of the

various polymorphic foms, narnely sub-a, a, 6, P' and B, of the b i n q mixture of POS

and SOS. As well, results of kinetic isothermai solidification as a function of temperature

were displayed as time-temperature-transformation diagrarns and crystal morphology

maps (Rousset et al., 1 998). Understanding the molecular structure and phase behaviour

of POP, SOS and POS and their mixtures will hopefully aid in the study of cocoa butter

polymorphism.

Cocoa Butter Polymorphism

It has been known for many decades that cocoa butter has various melting points

depending both on compositional differences and processing conditions. Cocoa butter

polymorphism has been extensively studied but contradictions over the number of forms

and their characteristics still exist. As early as 1900, it was reported that cocoa butter

had various melting points. During the next twenty years studies were carried out that

showed that cocoa butter codd crystallize in a stable and at least one unstable form (as

reported by Vaeck, 1960). In the late 1920's three polymorphic forms of cocoa buner

were known to exist with melting temperatures of X 0 C , 29°C and 34°C (Abers, 1928).

More than twenty years later a study was conducted that descnbed cocoa butter as having

four polyrnorpfuc forms known as y, a, B' and P (as reported by Vaek, 1960) with

melting points at 17"C, 23OC, 28°C and 35°C. Later another form was identified with a

melting temperature of 33OC, indicating the existence of a fifth polymorph (Duck, 1958).

The development of X-ray techniques allowed for a more detailed analysis and me

determination of six polymorphic foms of cocoa butter (Wille and Lutton, 1966).

In 1966, a complete study of the polymorphic states in cocoa butter was

conducted and determined the existence of the following polymorphic forms in order of

increasing stability: 1 (sub-a or y), II (a), III, IV (P7), V (B) and VI (Wille and Lutton,

1966). In this study, cocoa butter was cooled to different temperatures and the

polymorphic f o m was determined using X-ray diffkaction. Form 1 (y) was f o n d to be

the initial state formed when cocoa butter was melted and subsequently crystallized at

temperatures lower than 0°C. This form was shown to have a very iow order of stability

with a melting temperature of 173°C and shon spacings at 3.7A and 4.2A. Form II was

obtained when melted cocoa butter was quickly frozen and stored between several

minutes and an h o u at 0°C. Form II (a) was found to crystallize directly fiom the melt

or via a polymorphic transformation fiom form 1. F o m II was found to be fairly unstable

and at low temperatures (0°C and 5°C) transformed into form III. At Z 6OC and 21 OC

form II transformed to form IV. Form II was found to have a strong diffraction peak at

4.24A and a melting temperature of 23.3"C (Wille and Lutton, 1966). Form III (Pzy) was

obtained by meking the cocoa butter and then storing it at 5°C to 10°C or by

transformation of form II by storage at 5°C to 10°C. This form displayed moderate

stability with short spacings at 3.86A and 4.25A and a melting temperature of 25S°C.

Next, form IV (p 1 ') was obtained by crystallizing the melt at 16OC to 2 1 OC or by

transfomation of a lower melting state by storage at 16°C to 2 1 OC. This form, with a

melting temperature of 27S0C and short spacings at 4.1 SA and 4.35A, was fairly stable

however, d e r a certain time it transformed to forrn V at every temperature. F o m V (PZ)

was found to crystallize directly fiom the melt or fiom lower melting polymorphs. This

form remained stable for a very long period of time at the proper storage temperature

(Wille and Lutton, 1966). Form V was found to have a melting temperature of 33.8"C

and nine short spacings the strongest found at 3.98A and 4.58A. Finally, fom VI (Pz)

could not be obtained directly fiom the rnelt, but oniy by transformation of form V d e r a

long storage period at high temperatures. This crystal form was found to be the most

stable with a rnelting point of 36.3"C. However, there were no significant changes in the

shoa spacings as cornpared to form V (Wille and Lutton, 1966).

XRD data collected for the polyrnorphic forms observed by Wille and Lutton

(1966) have been confirmed by other research groups (Chapman, 1971 ; Lovegren et al.,

1976a; Lovegren et al. 1976b; Witzel and Becker, 1969). These six foms have also been

confïrmed using DSC (Chapman, 1971 ;Huyghebaert and Hendrichc, 197 1 ; Lovegren et

al., 1 976a: Lovegren et al., 1976b). Chapman (1 97 1) however, found the melting

temperatures of each form to be several degrees lower than previously reported. Electron

microscopy has also been used to observe the various polymorphic forms of cocoa butter

and different morphologies for al1 of the six forms were determined. These polyrnorphic

forms were also characterized by XRD (as reported by Berger et al., 1979).

As previously described, TAGs can pack in double- or triple-chain-length

structures. It is however quite unusual for a natural fat to crystallize in the triple-chah-

length structure. In order for this to occur nearly al1 of the TAGs must be similar to 2-

oleo-distearin as is the case for cocoa butter (Hagemann, 1988). The charactenstic long

spacings deterrnined for the polyrnorphic forms of cûcoa butter allow for the

characterization of the chah length structure. Forrns II, III and IV were found to have

long spacings ranging fiom 22.1A to 55.1A suggesting a double-chain-length structure

(Chapman, 197 1 ; Wille and Lutton, 1966; Witzel and Becker, 1969). Long spacings

ranging from 63.0A to 66.0A were determined for forms V and VI suggesting a shift to a

triple-chain-length structure (Chapman, 1971 ; Wille and Lutton, 1966; Witzel and

Becker, 1 969). In the case of the 2-oleo-disaturated TAGs found in cocoa butter the

triple-chain-length structure consists of Iayers of unsaturated chains with the sarne

structure whereas the saturated chains are packed in the triclinic packing as in the P foxm

of simple TAGs (Larsson, 1972).

Even though six distinctive polymorphic forms of cocoa butter have been

observed by several research groups, others have recognized only four f o m s (Merken

and Vaeck, 1980; Vaeck, 1960; as reported by van Maissen, 1996a). It has been

proposed that form III is merely a mixture of variable proportions of forms II and IV.

Form III has never been isolated in its pure state and its formation conditions are quite

similar to that of fonn N (Merken and Vaeck, 1980). Schlichter-Arnhime et ai. (1 988)

reported that cocoa butter does not behave differently than a pure triacylglycerol and

consists mainly of three possible polymorphic forms. The fact that six polymorphs have

been detected is related to the fact that complex mixtures of saturated and unsaturated

triacylglycerols are present. Therefore, form III c m be interpreted as being a mixture of

two solid phases of polymorph II and IV (Schlichter-Arnhime et al., 1988). However,

some researchers rnay not have observed form III because the samples were not tempered

in order to develop a particular form and only one sarnple of cocoa butter was used

(Timrns, 1984). It has been suggested that form VI is not a distinctive polyrnorph but is

identical to form V only lacking the liquid triacylglycero! fraction. Form VI was

determined to be sirnply a separation of the solid solution form V into two distinct phases

(Merken and Vaeck, 1980). It is possible that Merken and Vaeck (1980) were unable to

detect form VI because they failed to provide the long tempering time required for its

formation as shown in previous studies.

The determination of the various polymorphic forms of cocoa butter is one of the

most controversial areas in confectionery science. There is not only a discrepancy in the

nurnber of polymorphic forms but also in the nomenclature used to classifi these various

forrns. TabIe 2 i1lustrates the different nurnber of polymorphs thought to exist and the

nomenclature used by various researchers to describe these forms.

-- -

Table 2: Polymorphic forms of cocoa butter as detemined by various research groups

Wille and Lutton (1 966) Merken and Vaeck (1980) as reported by van Malssen et al. (1996a)

Polymorphic Tm (OC) Polymorphic Tm (OC) Polymorphic Tm (OC) F o m Form Form

In the past few years, confectionary fat research has focused more on the

transformations between polymorphic forms and the effect of processing conditions on

polyrnorphism. An in-situ processing ce11 was used to simulate the tempering and

shearing process of cocoa butter during chocolate manufacture, while collecting

synchrotron radiation data. Upon, cooling fiom 50°C to lS°C, cocoa butter was found to

crystallize at 20.g°C probably resulting in form IV. After reheating the sample to 28.7OC

a melting temperature of 33.8"C was observed indicating a polymorphic transition to

form V. This experiment allowed for the determination of the polyrnorphic forms present

during the vaious stages of chocolate tempering (van Gelder et al., 1996).

Loisel et al. (1998) used a new instrument, which allowed simultaneous DSC and

XRD recordings from the same sample in order to characterize intermediate phase

transitions. This confrrned the existence of six polyrnorphic forrns. It was proposed that

the structure of form 1 was a liquid-crystal organization in which some of the chains

displayed a P' crystal organization and others remained unordered. As well, fonn III was

oniy observed in a sharp temperature domain through its specific short-spacings (Loisel

et al., 1998).

Many of the previous studies have failed to indicate the cooling rate of the cocoa

butter during solidification in a qiimtitative way making it difficult to compare the

conflicting experimental results. Real-time XrCD investigations have been used to carry

out temperature-dependent crystallization experiments (van Malssen et al., 1996a; van

Malssen et al., 1996b; van Malssen et al., 1996~; van Malssen et al., 1999). The

polymorphism of cocoa butter was found to be determined mainly by the solidification

temperature, provided that this temperature was arrived at quickiy enough to prevent the

formation of higher melting polymorphs (van Malssen et al., 1996a). The y, a and B'

polymorphs were found to crystallize directly from the melt, whereas the P polyrnorph

was obtained only via a phase transformation from the P' form. Wide ranges of melting

temperatures were determined for the polyrnorphic forms unlike the specific melting

points reported in previous literature (as reported by van Malssen et al., 1996a). The

crystallization of the y phase was found to take place within seconds when the

temperature is less than 7°C and was always preceded by sorne a crystallization. The y

polymorph was found to have a melting range fkom -5°C to SOC, with short spacings at

3.7A and 4.2A. The formation of the a polymorph took place between 3OC and 22°C

with a cooling rate of at least 2°C per minute without the presence of more stable

polyrnorphic forms. This form was observed to have a XRD peak at 4.2A and a melting

range fiom 17°C to 22°C. At temperatures above 20°C, substantial parts of the sample

were found to be liquid. The P' phase was fornled from the melt at 24°C and 26°C

regardless of cooling rate with strong difiaction peaks at 4.2A and 4.3A and a medium

peak at 3 . X The melting range of the fl' polyrnorph was found to be frorn 20°C to

27°C. The P' phase can be formed much more quickly fiom the a polymorph than from

the melt (van Malssen et al., 1996a). No independent phase III or IV could be observed

in the formation of the P' polyrnorph. The formation of the P phase was observed only as

a transformation fiom the p' polymorph. The stable P polymorph was found to have a

strong short spacing at 4.6A and a group of short spacings between 3 SA and 4.0A with a

melting range of 39°C to 34°C (van Maissen et al., 1996a).

The diffraction patterns of the P phase were very similar to those of form VI as

determined by Wille and Lutton (1966), while the melting points corresponded with form

V. Kowever, the melting point of form VI as reported by Chapman (1 97 l), does agree

with these resuits (van Malssen et al., 1996a). The wide range of rnelting points and

small differences in diffraction patterns led to the conclusion that phase V and VI are hvo

subphases of the B polymorph. The j3 form was found to crystallize directly from the

melt at 2S°C due to a memory effect. Two different stages were found to occur, referred

to as short-term and long-term memory effects. In this study the cocoa butter was not

heated to a high enough temperature in order to destroy the crystd lattice structure

resulting in a memory effect and the direct formation of the P polyrnorph (van Malssen et

al., 1996~).

Van Malssen et al. (1 999) constnicted an isothemal phase-transition scheme for

cocoa butter under static conditions using their previous results as a guide. The data used

for th is phase diagram was mainly obtained by time resolved XRD. The y polyrnorph

was found to form directly fiom the melt and transform into the a polymorph or into the

a form and then the p' polymorph. The a polymorph formed easily either via a

transformation fiom the unstable y form or directly from the melt. The P' form was

considered to exist as a phase range rather than as two separate polymorphs as reported

previously (Wille and Lutton, 1966). The B' polyrnorph formed either via a

transformation fiom the a polymorph or directly fiom the melt. Lastly, the P polymorph

was formed ody via a phase transformation from the P' form (van Malssen et al., 1999).

This in-depth study of the phase behaviour of cocoa butter is of great importance because

it may help to optimize production processes and to rnaintain product quality.

Crys tallization of Fats

The study of fat crystallization processes allows for an understanding of their

physical and chemical properties. Above the melting point of a fat the molecules are in

random thermal motion and are constantly associating and dissociating with each other.

As the temperature is decreased the molecules tend to corne together and form clusters.

Once these clusters reach a certain size with a critical radius they are able to form a stable

nucleus (Garside, 1987; Lawler and Dimick, 1998; Larsson, 1994; Sato, 1989). The

critical value for the radius depends on the surface energy and the fkee energy of the

liquid to solid transfomation. In the formation of a nucleus there are two opposing

forces. Nucleation is favoured since energy is released fiorn the aggregating molecules;

however, the creation of a solid-liquid interface requires energy input to overcome

surface tension. A stable nucleus will form only when the heat of crystallization exceeds

th.e surface energy (Lawler and Dirnick 1998; Timms, 1995). In TAGs, the radius is not

an adequate measure of the size of a nucleus and it is more realistic to imagine that

crystailine dimers are aligned laterally in bilayer units of a certain size (Larsson, 1994).

There are three types of nucleation that occur in a fat system. Hornogeneous

nucleation occurs in a pure system and requires a very high driving force since there are

no foreign particle surfaces to induce molecular aggregation. Heterogeneous nucleation

occurs in a system that contains impurities. Homogeneous and heterogeneous nucleation

are referred to as primary nucleation (Garside, 1987; Larsson, 1994; Sato, 1989).

Secondary nucleation occurs once crystals have formed by primary nucleation. It results

from the fiacturing of growing crystals into smaller stable crystal nuclei. If the small

pieces of crystals that are removed are smaller than the critical size, they redissolve. If

Iarger, they act as nuclei and grow to become crystals (Timms, 1995). Prirnary

nucleation is usually heterogeneous since even in pure systems once fat crystals have

formed they act as impurities for further nucleation. Most natural fats, including cocoa

butter, contain sufficient irnpurities for heterogeneous nucleation to take place even at a

few degrees below the melting temperature (Walstra, 1987). Nucleation often occurs in

the a fom. However, at temperatures above the final melting point of the a polymorph

nucleation may occur in another polyrnorphic fom, but at a much slower rate (Walstra,

1987).

Once the crystals formed exceed a certain size they flocculate as a result of net

Van der Waals' attraction between them and little repulsion except at atomic distances

(Walstra, 1987). Crystal growth occurs when a crystallizing molecule d i f i ses ont0 the

growing crystal surface. When the driving force is very small (low degree of

supercooling), spiral growth occurs and when a greater driving force @gh degree of

supercooling) is present, two-dimensional growth occurs (Sato, 1989). The rate-

determining step is the incorporation of the new molecule in the correct configuration at

the correct place on the growing crystal surface (Garside, 1987; van den Temple, 1968).

The rate of crystal growth is proportional to the degree of supercooling and varies

inversely with viscosity since molecular diffusion is reduced as melt viscosity increases.

As the crystallization temperature is decreased, the viscosity increases and the growth

rate reaches a maximum and then decreases with increasing supercooling (Timms, 1995).

Greater supercooling results in &ter crystallization with more crystal faults since the

molecules fkom the melt attach to the crystal surface and have insufficient time to become

optimally arranged before new attachrnents are made. This results in a

thermodynamically unstable a metaphase formed due to kinetic preference. Kinetically,

the least stable forrn is favoured due to the lower difference in the surface fiee energy

between the melt and the unstable crystal surface (Boistelle, 1988; Timms, 1995). At

lower degrees of supercooling, the incorporation of new molecules will be more likely to

occur in the optima1 configuration because molecules will have time to orient themselves

correctly (Timms, 1995). As a result, the fat crystallizes into a more stable P' or P

polyrnorph (Walstra, 1987). As crystal growth proceeds, there is a supersaturation of

crystals, which causes an increase in the critical crystal size. This results in the growth of

larger crystals at the expense of smaller crystals (LawIer and Dimick, 1998).

Fat crystallization is generally a very slow process for a nurnber of reasons. The

triacylglycerol molecde has thee flexible fatty acid chahs that are difficult to fit into a

crystal lattice. During TAG crystallization there may be competitive inhibition by other

substances such as mono- and diglycerides (Walstra, 1987). Also, one crystal lattice

incorporates many different triacylgIycero1 molecules, some of which do not fit well and

delay the incorporation of the perfectly fitting ones. Finally, once crystals are formed, it

becomes much more difficult to remove heat fiom the fat due to its increased viscosity

(Walstra, 1987).

In complex fat systems, crystallization may never be complete because

thermodynarnic equilibrium is never attained and rearrangements of rno lecules are

constantly taking place. Over tirne, crystals may change in composition, usually

segregating into purer ones. Unstable a and P' polyrnorphs are transformed into the

more stable j3' and P forms (Walstra, 1987). Polymorphic transfomations fiom the least

to most stable forms, as a function of time and incubation temperature, are irreversible.

Transformations rnay occur either through the solid state or via the melt. Solid state

transitions may occur as a function of time at a particular temperature. Melt-mediated

transformation involves the process of melting the less stable form, nucleation and

growth of the more stable form and m a s transfer in the liquid by melting of the less

stable form. However, the rate of melt-mediated transformation is higher than

polymorphic transformations occumng in the solid state (Sato, 1 996).

Many have quantified the crystallization kinetics of fat systems using the Avrarni

equation (Dibildox-Alvarado and Toro-Vazquez, 1997; Herrera and Marques Rocha,

1996; Herrera et d., 1999; Kawamura, 1979; Metin and Hartel, 1998; Ng and Oh, 1994;

Wright et al., 2000; Ziegleder, 1990). The Avrarni equation was originally developed to

study the kinetics of the overall crystallization process of low rnolecular weight materials

such as metais (Avrafni, 1939; Avrarni, 1940; Awami, 1941). This theory was then

extended to the crystallization of polymers (Mandelkem et al., 1 954; Mandellcern, 1956).

The mechanism of nucleation and crystal growth has also been studied with poly-

ethylene succinate, poly-ethylene adipate, poly-ethylene oxide and isotactic

polypropylene using the Avrarni theory (Barnes et al., 1961). The Avrami mode1 can be

used to quantifi crystallization kinetics and describes the overall crystallization process

taking into accomt both nucleation and crystal growth (Awami, 1939; Awami, 1940;

Avrami, 194 1):

SFC(t ) -ktn = l - e

SFC@ )

where SFC (t) describes the solid fat content as a function of time, SFC (a) is the limiting

SFC as time approaches infinity, k is the Avrarni constant and n the Avrarni exponent.

The A m i constant k is a measure of the velocity of reaction, and shows an Arrhenius

type dependence on the crystallization temperature (Christian, 1965; Graydon, 1994).

The Avrarni exponent (n) describes the mechanism of crystallization. This pararneter is

sensitive to both the tirne dependence of nucleation and to the dimensionality of growth.

Nucleation can either be sporadic or instantaneous and crystal growth may occur in one,

two or three dimensions to give rods, discs or sphemlites, respectively (Sharples, 1966).

The different combinations of nucleation and growth mechanisms characteristic of the

Avrami exponent are shown in Table 3 (Sharples, 1966). A single value of n may

describe different types of nucleation, growth modes and is a combination of both of

these processes.

Table 3 : Avrami exponent values for the dif5erent types of growth and nucleation (Sharples, 1966)

Avrami Exponent Various types of growth and nucleation 3 + 1 = 4 Spherulitic growth fiom spor~dic nuclei 3 + 0 = 3 ~iherulitic growth fiom inçtantaneous nuclei 2 + 1 = 3 Disc-like growth fiom sporadic nuclei 2 + 0 = 2 Disc-like growth from instantaneous nuclei l + l = 2 Rod-like &owth fiom sporadic nuclei l + 0 = 1 Rod-like growth fiom instantaneous nuclei

Microstructure

Various techniques have been used to study the microstructure of food products

including fats (Vaughan, 1979). Electron microscopy (EM) has been widely used to

study the morphology of fats and food products (Brooker 1990; Buchheim, 1982; deMan

1982; Heertje et al., l987a; Kalab, 1983; Sargent, 1988). More recently confocal

scanning light microscopy (CSLM) (Heertje et al., 1 987b: Herrera and Hartel, 2000) and

multiple photon rnicroscopy (Marangoni and Hartel, 1998) have been used to study fat

systems. CSLM has the advantage over light microscopy and EM in that optical

sectioning can be performed without disturbance to the three-dimensional intemal

structure of the fat sarnpies.

Polarized Iight microscopy (PLM) is a well-established and increasingly used

technique for studying the relationship between microstructure, physical properties and

processing behaviour (Hoerr, 1960, Kellens et al., 1992; Manning and Dirnick, 1985;

Narine and Marangoni, 1999a; Rousset and Rappaz, 1996; Rousseau et al., 1996b). This

technique distinguishes between the solid and liquid phase because the crystals of the

solid phase are anisotropic and therefore birefiingent, whereas the liquid phase is

isotropic (Chawla and deMan, 1 990). As early as 1960, Hoerr descnbed the established

polymorphic forms using PLM. The crystals of the a form were found to appear as

platelets of about 5 urn. The P' crystals were described as small needles not exceeding 1

urn in length and the P crystds were found to be large ranging in size from 20 to 100 um,

often growing in clunps (Hoen, 1960). H~wever, microscopy alone should not be used

to identie polymorphic forrns, this technique should be carried out in conjunction with

XRD (Berger et al., 1979). PLM has also been used to illustrate the transformation of the

a polymorph to the f3' and fmally to the stable P form with increasing ternperature for

tripalmitin crystals (as reported by Berger et al., 1979).

Studies have been conducted to establish the relationship between the

microstmcture and polymorphism of cocoa butter and its major TAGs. PLM was used to

identie crystal morphologies and to measure the density and growth rate of the grains

(crystals) as a function of undercooling in POS, POP and SOS (Rousset and Rappaz,

1996). It was found that the morphologies of the grains were variable even for the same

triacylglycerol and polyrnorphic form (Rousset and Rappaz, 1996). This research group

also observed the difference in the microstructures of POS that were obtained by a-melt

mediated crystallization and direct crystallization. The latter gave a structure with large

spherulites, whereas a-melt mediated crystdlization resulted in very small fine crystals

even though both were in the P' form (Rousset and Rappaz, 1 997).

In 1932 (as reported by Vaeck, 1960), PLM was used to observe the morphology

of cocoa butter. In 195 1, Vaeck showed by PLM that cocoa butter is able to exist in at

least two modifications, the stable one growing slowly at the expense of the unstable

form. At O°C a bright crystalline mass was observed and when heated to room

temperature the morphology became much more dull. Mer a few hours at room

ternperature, very bright crystal nuclei were obsewed. These grew rapidly to form small

spherulites of approximately 50 pm in diameter. Afier 5 days of crystallization the

spherulites were so large that they occupied nearIy the entire field of view (as reported by

Vaeck, 1960). The stable f o m of cocoa butter was also observed to have a needle-like

morphology (as reported by Vaeck, 1960).

PLM and scanning electron microscopy (SEM) were used to chaïacterize the

crystalline siructures of cocoa butter after extensive storage at high temperatures (26°C to

34°C) (Manning and Dimick, 1985). These techniques were used in conjunction since

PLM represents a cross sectional (or internal) view of the fat crystal while SEM allows

for the characterization of an unrestricted fiee growing crystal (Manning and Dimick,

1985). A mesh of crystals appeared early on when cocoa butter was crystallized at 26 OC.

After the mesh was formed distinct feather crystal growth appeared on the periphery.

During this sarne time, the appearance of smaller yet distinct (individual) crystals were

observed thoughout the sarnple. After a few days of storage at 26 OC the extension of the

feather crystalline form was observed along with the melting of the individual crystals

and subsequent growth of a less distinct phase with a blade-like appearance (Manning

and Dimick, 1985).

At 28°C four different morphologies identified as mesh (temper), feather, spiney

and needle were found to exist depending on the storage tirne. At îO°C the morphology

was described as feather-like and spiney. Finally, at 32OC both irregular and spiney

crystals were found. Melting points for these different morphologies indicated that the

structures were either p' or p polyrnorphs. This led to the conclusion that the

polymorphic forms can be diverse in terms of visual structure and crystal appearance

(Manning and Dimick, 1985).

Manning and Dimick (1 985) also compared the microstructure of cocoa butter

with that of semi-sweet dark chocolate. The chocolate was incubated under the sarne

conditions as the pure cocoa butter. The crystals were found to appear very similar in

both cases. For both cocoa butter and chocolate, the changes during crystaliization were

shown to be quite variable. One degree change in incubation temperature was shown to

permanently change the crystal formation (Manning and Dimick, 1985).

Manning and Dirnick (1 985) also investigated the differences in composition of

the various crystd structures. At incubation temperatures fkom 26°C to 30°C, the initial

crystal formed during tempering was found to exbibit the properties of a high melting

point crystal seed. The stability of this seed was found to increase as the crystallization

temperature increased. At this stage, the composition of the initial crystal was not

positively identified but it was believed to be an SOS-nch fiaction. Later work done by

this group did determine compositional differences of the crystals formed throughout the

incubation at various crystallization temperatures (Dimick and Manning, 1987). At 26"C,

no compositional differences were found for the original mesh crystal. However, the

feather-like and individual crystals showed significant increases in SOS and concurrent

decreases in POP. There was no significant difference in the POS content of these

crystals. It was concluded that various cocoa butter triacylglycerols act in a manner

characteristic of hctional crystaliization (Dimick and Manning, 1987).

The microstructure of cocoa butter has also been observed using transmission

electron microscopy (TEM) (Hicklin et al., 1985) where the morphofogy was detennined

for the six polymorphic forms of cocoa butter as defined by Wille and Lutton (1 966).

Correlations were made between the known molecdar structure as determined by XRD

and DSC, and the morphology observed in most of the polymorphs. However, in some

cases, knowledge of the polymorphic form did not enable an accurate prediction of

morphology (Hicklin et al., 1985).

The study of microstructure of fat systems has become increasingly important

since the functional properties of many foods depends on a knowledge of their fine

structure. Microstructure is dependent on the composition of a fat and the characteristics

of the microstructure in turn determine the physical properties of the fat. Polarized light

micrographs of hydrogenated canola oil (deMan, 1982) and tripalmitin (Kellens et al.,

1992) show that a particular polyrnorphic form can be associated with different

microstnictures depending on the processing conditions. It is possible to obtain an idea

of the shape and size distribution of crystals by PLM; however, until recently

rnicroscopic techniques were not quantitative. A study done in our laboratory

investigated the effect of chernical interesterification (CI) of butterfat-canola oil blends

on rheological properties (Rousseau et al., 1996~). They found the CI did not

substantially decrease the solid fat content or change the melting characteristics

(Rousseau et al., 1996a). XRD codirmed that the predominant crystal fom, both before

and after CI, was the B' form (Rousseau et al., 1996b). However, the rheological

properties were quite different (Rousseau et al., 1996~). From LM and SEM, the crystal

arrangement after CI was shown to change dramatically (Rousseau et al., 1996b). These

observations pointed to the fact that it was not the SFC andor the crystal polymorphic

form responsible for the differences in the rheological properties of the CI and non-CI

sarnpIes, but in fact the macroscopic structure of the network of fat crystals in liquid oil.

The differences in the fat crystal networks of the CI and non-CI were characterized using

fiactal geometry (Marangoni and Rousseau, 1996). Both the concept of fractal geometry

and the development of a mode1 for fat crystal network characterization are described in

the next section.

Fractals

A new geometry was developed in the seventies, to describe many of the irregular

and fiagmented patterns found in nature. This new family of geometrical shapes was

given the name fractals (Mandelbrot, 1983). These shapes are not lines, planes or 3-

dimensional objects and therefore cannot be explained using Euclidean geometry. Fractal

geometry is concerned with the geometric scaling relationships and syrnrnetries

associated with fiactal objects. An important charactenstic of perfect fiactal objects is

that they are self-similar at al1 levels of magnification. A fractal system can display

statistical self-similarity rather than exact self-similarity, where the microstructure is

similar over a certain range of magnification (Meakin, 1988). The fiactal geometry

pnnciples can also be used to describe a disordered distribution of mass including

particles in a colloid gel and fat crystal networks. In this case, at diEerent scales of

observation the patterns are statistically self-similar and the relationship of the radius (R)

to the mass (M) is given by:

M(R) - R~ (2)

where D is the mass fiactal dimension which is also referred to as the fractal dimension

(Marangoni and Rousseau, 1996; Meakin, 1988; Narine and Marangoni, 1999a; Vreeker

et al., 1992b). If this equation were describing a two-dimensional Euclidean object, like a

square, then the value of D would be 2. However, a fiactal object may be classified as an

intermediate between a line and a plane or a plane and a cube. Therefore, the fiactal

dimension w-OUM be fiactional with a value between 1 and 2 or 2 and 3 (Narine and

Marangoni, 1999d). Fractal geometry has been well reviewed by Jullien and Botet

(1987) and Meakin (1988).

Afier the introduction of the fiactal concept many studies were carried out to

explain the structures of polymer and colloidal aggregates. The scaling theory has been

used to explain the elastic properties of protein gels (Bremer et al., 1989; de Gemes,

1979; Stading et al., 1993; Vreeker et al., 1992a) and colloidal aggregates have been

established as fractal structures both rheologically and optically (Ball, 1989; Brown and

Ball, 1985; Buscall et al., 1988; S o ~ t a g and Russel, 1987; Uriev and Ladyzhinsky, 1996;

Weitz and Oliveria; 1984). From work with colloidal aggregates a power-law

relationship of the elastic modulus to the solid volume fiaction was established (Ball,

1989; Brown and Ball, 1985; Shih et al., 1990; Somtag and Russel, 1987; BuscalI et al.,

1988).

The scaling behaviour of the elastic properties of collcidal gels was studied by

Shih et al. (1990). They developed a scaling theory based on the structure of the gel

network as a collection of flocs that are fiactal objects closely packed throughout the

sample. Two regimes, the strong-link and the weak-link, were identified based on the

strength of the links between the flocs relative to the strength of the links within the flocs

thernselves. When a network is composed of very large flocs, which occurs at low

particle concentrations (low SFC), the links between the flocs are stronger than the Bocs

themselves. For this strong-link regime, the elastic constant is related to the solids

volume fiaction as:

The macroscopic elastic constant (K) hcreases with particle concentration (9) in a power

law function with the exponent (d-x)/(d-D) (Shih et al., 1990)). The Euclidean dimension

d is usually 3, D is the fractal dimension, and the backbone fractai dimension (x) usually

lies constant between 1 and 1.3 (Shih et al., 1990). When a network is composed of very

small flocs formed at a high particle concentration (high SFC), the links between the

flocs are weaker than the flocs themselves. For this weak-link regime, the elastic

constant is related to the solids volume fiaction as:

K- <p(d-2"d-D'

Once again, this equation shows that the elastic constant increases as a function of

particle concentration in a power law function but this time with the exponent (d-2)/(d-D)

(Shih et al., 1990). Equations 3 and 4 show that in the weak link regime the elastic

. constant of the system increases more gradually with particle concentration than in the

strong-link regime (Shih et al., 1990).

The mechanical properties of fat crystal networks were first explained in terms of

a simple network mode1 (van den Tempel, 196 1). Van den Tempel proposed that fat

crystal networks are composed of straight chains of fat particles held together by

attractive van der Waals forces. It was Iater determined that, in reality, the network is

built fiom aggregates of fat particles and not of individual crystals. A quantitative

description of fat crystal networks using the fiactal concept was first attempted in 1992

by Vreeker and CO-workers (Vreeker et al., 1992b). Rheological data was analyzed using

the scaling theories developed for colloidal gels. The elastic modulus was shown to have

a power-law dependence on the particle volume fiaction as predicted by the colloidal gel

models. A fiactal dimension was calculated for the fat network using the strong-link

41

regime described above. This work also describes the determination of a fiactal

dimension for the crystal network uskg light scattering methods. These agreed with

values calculated fiom the strong-link equation (Vreeker et ai., 1 992b). This research

however, did not demonstrate that fat crystal networks at low particle volume fractions

are organized in a similar way to colloidal gels. Although they did not justifj the use of

the strong-link regime theory, the relationships established in this work remain very

important in the study of fat crystal networks.

Fractal geometry was also used to characterize the structure of the fat crystal

network in milkfatlcanola oil blends in order to detennine the effects of interesterification

GE) on the structure of the plastic fat (Marangoni and Rousseau, 1996). The weak-link

mode1 for high concentration colloidal gels was developed for fat crystal networks of

hi& solid fat content. The rheological application of this theory demonstrated a power-

law relationship between the shear elastic moduli (G') of the networks and the solid fat

content of the network given by:

G'- (SFC)"

The exponent m represents the slope used to derive the fiactal dimension assuming the

weak-link regime by:

m = (d-2)/(d-D) (6)

IE was found to cause a decrease in the fractal dimension. It was proposed that this large

change in the structure of the fat crystal aggregate network could be responsible for the

drastic decrease in the observed hardness (Marangoni and Rousseau, 1996). These

researchers also studied the effect of interesterification on blends of lard/canola oil and

palm oil/soybean oil (Marangoni and Rousseau, 1 998a; Marangoni and Rousseau,

1998b). Once again a power-law relattionship between the elastic modulus (Gy) and the

SFC was established:

G'= Y(SFC)~ (7)

where y can be related to the properties of the particles that make up the network

(Marangoni and Rousseau, 1998b). In this work, it was found that the fiactal dimension

did not change upon IE for both the lard and palm oil systems but the factor y increased

fourfold in the IE lard system. The factor y did not change significantly for the palm oil

system (Marangoni and Rousseau, 1998b). After examining polarized light images for

these fat systems, it was suggested that the rheological properties of palm oil and lard are

controlled by the properties of the particles that make up the network. Accoràing to the

work done by this research group, the scaling behaviour of the fat crystal network

according to the weak link regime seems to suggest that there are three important

indicators of macroscopic hardness: solid fat content, fiactal dimension and the factor y

(Marangoni and Rousseau, 1998b).

The fractal dimension of a fat crystal network has also been determined from

PLM images. An analytical technique to quantifi the microstructural level of the fat

network was established by Narine and Marangoni (1999a). In this method the number

of particles present in a three-dimensional portion of the sample were determined by first

representing d l of the particles present in that portion of the sarnple in the plane of the

image. D was related to the number of particles (N), to the linear size of the fkactal (R)

and to the linear size of one particle (microstructural element) (c):

N = (R/clD (8)

It was then assumed that the size of the microstructural element was statistically constant

resdting in:

N = R ~ (9)

Then by taking logarithms:

N(R) = logloc + DlogioR (10)

where c is a constant and N(R) is the number of particles in a cube length R in the image

(Narine and Marangoni, 1999a). The number of particles for various values of R are then

determined and loglo N(R) is plotted as a function of logio(R) with the resulting dope

giving the fractal dimension. The fiactal dimension calculated using this image analysis

technique and those calculated by rheological methods as described earlier were shown to

have excellent agreement (Narine and Marangoni, 1999a). It was demonstrated that

systems with a higher fiactal dimension have a greater degree of order in the packing of

the microstructurd elements (particles (Figure 1)) within the rnicrostnictures (clusters

(Figure 1)) than those systems with lower fractal dimensions (Narine and Marangoni,

1 999a).

The fractal nature of cocoa butter has recently been investigated. Narine and

Marangoni (1 999c) studied the physical properties of cocoa butter and SalatrimO. Both

of these fats were found to have almost identical melting profiles. As well there were no

significant differences in the solid fat content of the two fat systems at various

crystallization temperatures. However, PLM images showed striking differences in the

crystal network characteristics. Cocoa butter was found to be composed of large

microstnictures with a dense core surrounded by less tightly packed crystalline materid.

SalatrimB, on the other hand was observed to have a microstmcture of small randomly

arranged non-crystalIine translucent platelets. The differences between these two

systems were explained by differences in triacylglycerol composition and polyrnorphism.

The microstructure of SalatrimB could not be quantified because it is a random structure

so the fkactal theory cannot be applied (Narine and Marangoni, 1999~). When

characterizing the spatial distribution of mass using fiactal dimensions, one must be

confident that the system is actually fiactal. In th is particular study, the microstructure of

the cocoa butter was quantified and found to have a fiactal dimension of 2.3 1 according

to image analysis. Rheologically the fiactal dimension was detemined to be 2.37. This

shows that there is a fairly good agreement between the fiactal dimension obtained

rheologicdly and microscopically (Narine and Marangoni, 1999~). In most cases a

higher fiactal dimension c m be related to a greater elastic modulus (G') value but this is

also dependent on the constant y. Cornpared to other fat systems observed by Narine and

Marangoni (1999a), cocoa butter was found to have a lower fiactal dimension than palm

oil, lard and tallow but greater than milkfat. Tt has also been speculated that processing

conditions will have an effect on the fiactal dimension (Narine and Marangoni, 1999a).

OBJECTIVES

Cocoa butter polyrnorphism and its influence on final product quality has been

extensively studied. However, the effect of crystallization kinetics and microsûuctural

characteristics on sensory attributes have been overlooked. The aim of this work is to

sfudy the reIationship between crystalIization behaviour and microstructure.

1) The main objective of this work is to estabhsh a relationship between

polyrnorphism, crystallization behaviour and microstmcture of statically crystallized

cocoa butter. As well, the influence of processing conditions on these various Ievels of

structure will also be studied.

2) The polymorphic forms and transformations of statically crystallized refined

cocoa butter will be investigated utilizing melting temperature ranges as established by

van Malssen et al. (1999) and published characteristic short spacings as determined by X-

ray difiaction. A time-temperature state diagrarn will be constnicted, which will be used

as a guide in order to investigate the other levels of structure.

3) Isothermal crystallization behaviour will be studied at various degrees of

supercooling. Crystallization kinetics will then be quantified in order to describe the

overd1 crystallization process including the tirne dependence of nucleation and the

dimensionality of growth. The different crystallization mechanisms will then be related

to poiymorphisrn and microstructural characteristics of the network.

4) Polarized light microscopy (PLM) will be used to image the microstructure of

statically crystallized cocoa butter. The rnorphology of the various polyrnorphic forms,

as established by the state diagram, and obtained through different processing conditions

will be determined. Microstructurd changes associated with polyrnorphic

transformations as a f i c t i o n of time will also be established.

5 ) The spatial distribution of mass within the fat crystal network will be quantified

using the concept of fiactal geometry (Narine and Marangoni, 1999a). This provides an

indication of the spatial dimension of mass of the network and is related to rheological

mechanical properties. Attempts will be made to relate the quantified microstructure to

polyrnorphism and crystallization behaviour.

MATERIALS AND METHODS Source Materials

Refined cocoa butter was obtained fiom Cacao de Zaan (Koog aan de Zaan, The

Netherlands) .

Triacylglycerol Profile

Cocoa butter TAG composition (carbon number (C)) was determined by gas-

liquid chromatography (GLC) (Shimadm GC-8A (Tokyo, Japan)). A sample of 100 mg

of melted sample in 500 PL of iso-octane (Fisher Scientific) was prepared and 1 pL was

injected into the GLC at 360°C. Runs were performed fi-om 270°C to 340°C at SOC per

minute and the flarne ionization detector was set at 360°C. A 0.7 m glass column was

packed with 3% OV-1 on Chromasorb W, HP 80110 mesh (Supelco Canada, Mississauga,

ON) with high purity nitrogen as the carrier gas (BOC gases, Guelph, ON). The flow

rates were 60 ml/min, 50 mL/min and 500 mllmin for nitrogen, hydrogen and air,

respectively .

Fatty Acid Profile

Free fatty acid profiles were deterrnined using the methylation method of Bannon

et al. (1985). A 50 mg sample of melted cocoa butter was dissolved in 2 ml of iso-octane

(Fisher Scientific). Then 200 pL of 2N potassium hydroxide (Fisher Scientific) in

methanol (Fisher Scientific) was added and the mixture was vortexed for 1 minute. M e r

5 minutes, 2 drops of methyl orange (Fisher Scientific) and ZN HCI (Fisher Scientific)

was added d l a pi& endpoint was obtained. Then 0.5 pL of the fatty acid methyl .

esters was injected into a gas-liquid chromatograph (Shimadzu GC-SA (Tokyo, Japan)).

A 1.5 m glass column with a 5 mm outer diarneter packed with Silar 9CP on Chromosorb

W (801100 mesh) (Chromatographie Specialties, Brockville, ON) was used. Runs were

performed from 60°C to 2 10°C at 6°C per minute. High purity nitrogen was the carrier

gas @OC gases, Guelph, ON) and the flame ionization detector and injector were set at

230°C. The flow rates were 15 mL/rnin, 50 mL/min and 500 d / m i n for nitrogen

hydrogen and air respectively.

Fatty Acid Content

The AOCS official rnethod Ca Sa-40 was used for determination of total fkee fatty

acids (FFA). A 28.2 + 0.2 g sarnple of melted cocoa butter, 50 mL of hot neutralized

ethyl alcohol (95%, Commercial Alcohols, Inc., Brampton, ON) and 2 mL of

phenolphthalein (Fisher Scientific, Nepean, ON) were constantly mixed in an erlenmeyer

flask at 40°C. This mixture was then titrated with 0.1 N sodium hydroxide until the first

permanent pink colour of the sarne intensity as that of the neutralized alcohol before the

addition of the cocoa butter was observed. The titration was determined to be compIete

when this colour persisted for at least 30 seconds. In most fats, including cocoa butter,

the percentage of fatty acids is calculated as oleic acid:

ml of aikali x N x 282 Free fatty acids as oleic, % =

Wt. of sample

Phosphorous Content

A method developed by Laboratory Services (Guelph, ON) was used in order to

determine phosphate content. A 2.5g sample of cocoa butter was weighed into a 250 mL

flask and 5 mL of concentrated sulfûric acid was added. The flask was then placed on a

hotplate at high temperature for 1 hour and then slightly cooled before adding 4 to 6

drops of 30% hydrogen peroxide. The flask was then returned to the hot plate for 10

minutes. The addition of hydrogen peroxide and short heatingkooling periods were

repeated until the solution became clear and colourless. Then a few drops of hydrogen

peroxide were added and the flask was Iefi on die hot plate for 30 minutes. The flask was

then cooled to room temperature, filled to the 250 rnL mark with deionized water, sealed

and mixed by inversion and shaking. Undigested material was left to settle ovemight and

an aliquot was decanted into a test tube for analysis. The phosphate concentration (mg/L)

in the digestion solution was measured by a technicon auto andyzer (TAA). The percent

phosphorous in the solution was calculated as follows:

250rnl % in sample = conc. + 10000

Solid Fat Content Determination

The crystallization behaviour of cocoa butter was followed by meamring the

change in solid fat content (SFC) as a function of time. Glass NMR tubes were filled

with approximately 3 grams of melted cocoa butter and heated for 30 minutes at 80°C.

The samples were then placed directly in a water bath at the desired crystallization

temperature. SFC was determined by pulsed Nuclear Magnetic Resonance @NMR) with

a Bruker PC/20 series NMR Analyzer (Bruker, Milton, ON, Canada). SFC

rneasurements were taken a suitable time intervals at each crystallization temperature.

Two determinations of two replicates were performed.

Differential Scanning Calorimetry

The peak melting temperature of cocoa butter crystdlized under different

conditions was determined using differential scannulg calorimetry (DSC). Approximateiy

6 mg of melted cocoa butter was introduced into standard DSC aluminum pans and

hermetically sealed. Pans were then placed in glas viais and heated for 30 minutes at

80°C. The vials were capped and then placed on pre-chilied aluminurn trays in an

incubator at a predeterrnined crystallization temperature. Afier the desired crystallization

time, the pans were transferred to a Dupont Mode1 29 10 DSC (Wilrnington, DE). The

DSC was set to the sarne temperature as the pan being introduced and analyses were

performed fiom the crystallization temperature to 50°C at a heating rate of 5°C per

minute relative to an empty pan. Empty pans were used for the baseline calibration,

indium was used for the ce11 constant cali'oration and a two-point temperature calibration

was perfonned using indium and gallium. The DSC monitored the changes in heat flow

of the samples during melting and expressed these results as heat flow (Wlg) versus

temperature (OC). Therrnograrns were analyzed for temperature maxima. One

determination of each of four replicates was perforrned.

Powder X-Ray Diffraction

The polyrnorphic modifications of cocoa butter were determined by powder X-ray

difiaction O(RD). Approximately 50pL of melted cocoa butter were measured into ô

glass capillary tube and placed into a pre-chilled glass vial in an incubator set to the

appropriate crystallization temperature. At a pre-determined time, the polyrnorphic f o m

of the crystailized cocoa butter sample was detennined using an Enraf-Nonius Kappa

CCD diffractometer (Nonius, Delft, The Netherlands). The samples were mounted

horizontally in modeling clay and placed in the X-ray beam at the temperature at which

the sample was stored (rnaintained by circulating liquid nitrogen). The X-ray bearn was

generated by a Molybdenum anode set at 50 kV and 36 rnA. The bearn stop was al1 the

way out and the carnera distance was set at 165m.m. There was a 1" rotation on phi and

data was collected over 1 iteration for 5 minutes. The system was calibrated using

calcium sulfate, which has strong reflections at 13.6A, 4.27A, 3.80A and 2.80 A.

Polarized Light Microscopy

The microstructures of the various polporphic forms of cocoa butter were

observed using polarized light microscopy (PLM). One drop (-1 5 uL) of cocoa butter

heated for 30 minutes at 80°C was placed on a glass microscope slide pre-heated to 80°C.

A pre-heated coverslip was then placed on top of the sample. The coverslip was placed

parallel to the plane of the slide and centered on the drop of sample to ensure that the

sarnple diickness was uniform. The samples were then placed on pre-chilled metal pans

at the desired crystallization temperature. After a pre-detemined time, the slides were

observed under polarized light using an Olympus BH polarized light microscope

(Olyrnpus, Tokyo, Japan). Slides were kept at the desired temperature during the

imaging using an LTS 350 large heating and ffeezing stage operated by a TP93

temperature programmer (Linkam Scientific instruments Ltd, Surrey England). An

electric element was used to heat the stage while liquid nitrogen (BOC gases) was used as

the coolant. Images were recorded using a Sony XC-75 CCD video carnera (Sony

Corporation, Japan) with the gain switch in the auto position. The images were digitized

using Scion Image software (Scion Corporation, Fredrick, MD, USA). Image quality

was enhanced by taking the average of 16 fiames and by applying a background

correction, using the scion software. Three images were captured fiom each of four

slides at the desired time and temperature combination.

Image Processing and Particle Counting

Images were processed using Adobe Photoshop 5.5 (Adobe Systems inc., San

Jose, CA, USA). Original images were inverted and thresholded using the bilevel auto

threshold cornmand. In most cases the auto threshold provided a good representation of

the reflections present in the images. In a few of the images, where two different

microstructures were present, a manual threshold was used since the auto threshold was

not found to provide an adequate representation of the image. The particle counting

method (Narine and Marangoni, 1999a) was used to quanti@ the thresholded images. A

series of boxes of increasing size were layered over the image and the nurnber of

reflections in each box was determined (Figure 7A). Reflections of less than 10 pixels

and greater than 10 000 pixels were not counted. The number of reflections was then

plotted as a fûnction of the length of the box and the slope gives an estirnate of the fiactal

dimension (Figure 7B).

Statisticrtl Analysis

Means of peak melting temperatures were calculated for each at predetermined

crystallization temperature. These rneans were compared using the Tukey method (a =

0.05) to test for significant differences between specific means. A similar method was

used to determine significant differences between specific means as calculated for

Avrami exponents, fiactal dimensions and solid fat contents. The correlation of

determination (r2) was calculated in order to correlated the Avrami exponent with

induction time, induction time with fractal dimension and Awami exponent with fiactal

dimension.

Slope = D

log (Length of Box)

Figure 7: Particle counting involves the determination of the number of reflections for various box lengths (A). The fkactal dimension c m then be determined by plotting the number of reflections as a function of the length of the box (B).

RESULTS AND DISCUSSION Chernical Composition

The smallest level of characteristic structure present in fat crystal networks

is found at the molecular level. Cocoa butters fiom different regions may Vary

significantly in TAG composition leading to differences in crystallization behaviour and

hardness (Chaiseri and Dimick, 1989; Shukla, 1995). Tables 4 and 5 show the fatty acid

(FA) and triacylglycerol (TAG) compositions, respectively, of the cocoa butter used in

this study, as determined by gas-liquid chromatography (GLC).

Table 4: Fatty Acid Composition of Cocoa Butter Faîty Acid Cocoa Butter (wt %) 16:O 28.1 + 0.45a 18:O 34.4 I 0.47" 18: 1 32.6 t 0.28a 18:2 2.8 1 If: 0.06a 18:3 and 20:O 1.95 k 0.21a aData represents the average of seven replicates f standard error.

The TAG composition agrees well with previous studies of cocoa butters fiom

various regions (Table 5).

Table 5: Triacylglycerol Composition of Cocoa Butter Triacylglycerol (carbon Published Resultsa (wt%) Expenmental Results nurnber) 50

aReference (Young, 1 984) b ~ a t a represents the average of four replicates 2 standard error.

The cocoa butter used in our work was refined, a treatrnent that generally reduces

FFA content. Free fatty acids were found to represent 1.2% of the total weight (Table 6),

which is within the range of 0.88% to 1.46% reported by Dimick (1 999).

Table 6: Free Fatty Acid and Phospholipid Content of Cocoa Butter. Component Cocoa Butter Free fatty acids (wt %) 1.2 k O.OOa Phosphorous (wt%) < 0.006 a Data represents the average of three replicates f standard error.

Since a FFA content of 1.2% is in the middle of this range, it is difficult to speculate

whether this particular cocoa butter had a high FFA content before refining or whether a

substantial decrease did not occur during treatrnent. Refming usually removes the more

polar components of cocoa butter and analysis indicated our sample had a low

phospholipid content (Table 6). Using an average molecular weight of 775 mol,

(calculated using the average of palmitic, oleic and stearic acids as the lipid component

and phosphatidylcholine), a phospholipid content of less than 0.15% (wt %) was

detennined. Work done by Davis and Dirnick (1989) determined that unrefined cocoa

butter had a phospholipid content of approximately 0.37% (wt %). nierefore refining

resulted in a substantial decrease in the phospholipid content of our cocoa butter sampie.

It has been speculated that polar components play a significant role in crystal seed nuclei

formation (Dimick, 1999). Therefore, refined fat systems will most likely crystallize in a

different manner than unrefined ones. This may in turn lead to differences in physical

properties such as melting and hardness characteristics.

Polymorphism

The structure of the solid state is the next level of structure known to affect the

macroscopic properties of fats. This level of structure is particularly important for cocoa

butter as it has been shown to exhibit six polymorphic f o m s (see literature review section

Cocoa Butter Polymorphism).

In this study, the polymorphic forms of cocoa butter were detennined fiom the

peak melting temperature obtained by DSC, and confmed by powder XRD. After

melting cocoa butter sampres at 80°C and upon crystallization at specific temperatures for

determined lengths of time, DSC melting profiles were obtained (Figure 8). A heating

rate of 5°C per minute fiom the crystallization temperature to 50°C was used in al1 of the

DSC experiments. The predominant polymorphic form was determined fkom the peak

melting temperatures (Figure 8A) based on published melting ranges (Table 2) (as

reported by van Malssen et al., 1996a). Polymorphic transformations are referred to as

monotropic, in that they are irreversible fkom the least to most stable forms as a fimction

of time and temperature. In these transition regions, two peak melting temperatures

were observed.

Cocoa butter crystallized at 0°C for 3 days had a melting profile displaying two

peak temperatures (Figure 8B). The first peak at 19.7OC is characteristic of the a form

while the second peak at 24.4OC indicates that the more stable P' form was also present.

A polymorphic transition is not always indicated by the presence of two peak

temperatures in one melting profile. Often, some of the replicates measured at the same

time-temperature combination displayed a single peak temperature characteristic of a

single polymorph, whereas other replicates indicated the presence of two polymorphic

foms. Two sarnples measured after a 6 day incubation at 20°C displayed different

melting profiles (Figure 8C). The first had a peak temperature at 27.3OC while the other

Temperature ( O C ) Temperature (OC)

Temperature (OC) Temperature (OC)

Figure 8: Overiay of three characteristic melting profiles obtained by DSC of the different polymorphic forms of cocoa butter (A). Melting curves of cocoa butter statically crystallized at O OC for 3 days (B), 20 O C for 6 days (C) , and -20 OC for 2 days (D) are also shown.

showed a peak temperature of 3 1.8"C. Under these particular conditions, a polymorphic

transformation is likely taking place fiom the P' form to the more stable P polymorph.

The metastabfe y polyrnorph has been shown to form directly fiom the melt at low

crystallization temperatures (van Malssen et al., 1995a). In our DSC experirnents, the

existence of a pure y phase was not observed. However, a peak temperature of i .6"C,

which is within the reported melting range for the y polyrnorph, was shown to exist

concurrently with a peak temperature of 20.6"C charactenstic of the a form at low

temperatures (Figure 8D). The y form, with a peak melting temperature between -5°C

and 5"C, was not observed d e r 10 minutes nor after 1 day as would be expected lkom an

unstable polymorph. Rather, the DSC rnelting profiles indicated that the a polymorph

formed after 4 minutes and the presence of the y polyrnorph becarne evident following 2

days of crystallization at either -1 5°C or -20°C. Since polymorphic transformations are

monotropic and the y polyrnorph is less stable than the a form, the occurrence of a

polymorphic transition is unlikely. We speculate that fiactionation has taken place,

resulting in the separation of different tnacylglycerol species. Some of the lower melting

triacylglycerols could have then crystallized into the y form and coexisted with the a

phase even after 7 days at -1 SOC and -20°C. DSC did not allow for measurements until 3

or 4 minutes into the crystallization process. Since the y polymorph is very unstable, it is

possible that there had already been a polymorphic transition to the more stable a form

before the peak melting temperature could be determined.

To determine changes in polymorphism fiom DSC rneiting profiies, peak

temperatures were plotted as a function of crystallization temperature at various times.

Changes in peak melting temperature plotted as a function of time after 7 days of

crystallization are shown in Figure 9. Six statistically different regions were determined

(P<0.00 1) (Figure 9). The first region included crystallization temperatures kom -20°C

to -5"C, where peak melting temperatures ranged between 18.4"C and 20.7OCY

characteristic of the a polymorph. The next region included crystallization temperatures

between O°C and 1 O°C, with peak melting temperatures ranging fiom 19.7OC to 259°C.

Ln this region, some of the peak temperatures were characteristic of the a form while

others indicated the CO-existence of the a and B' polymorphs. The third statistically

significant region included crystallization temperatures between 2S°C and 20°C. The

peak meiting temperatures found in this region ranged fiom 23.1 OC to 27.2"C

characteristic of the P' polymorph. Crystallization temperatures overlap with those in

the previous region (0°C to 10°C) because of the transition from the a polymorph to the

p' polymorph. Many of the peak temperatures in these two regions are similar and both

indicated the existence of the P' form. The fourth region included crystallization

temperatures between 10°C and 22"C, with peak melting temperanires ranging fi-om

24.g°C to 28.3"C. These peak temperatures were higher than those in region three, but

are also characteristic of the p7 polymorph. Therefore, this analysis indicated that at least

two different P' forms were present, with slightly different peak melting temperatures.

Figure 9: Peak melting temperature as a function of crystallization temperature obtained fiom DSC melting profiles of cocoa butter statically crystallized for 7 days. Syrnbols represent the average k standard error of four replicates.

It is difficult to distinguish between these two foms since their peak melting

temperanires are so close. The fifth statistically significant region included

crystallization temperatures between 20°C and 25°C. Peak melting temperatures in this

region ranged from 26.7OC to 33.70C7 which is characteristic of the polymorphic

transition region fiom the p' form to the f3 polyrnorph. The final region including

crystallization temperatures ranging fkom 23OC to 26"C, with peak melting temperatures

ranging fiom 29.0°C to 33 .70C7 charactenstic of the f3 polymorph.

Similar results were obtained for cocoa butter crystallized statically at

temperatures ranging fiom -20°C to 26°C at al1 times. Our work suggests that peak

melting temperatures provide a good indication of the different polymorphic foms. The

various polymorphic regions discussed in this work known as a, a to P', B7, B' to P and B

were al1 found to be statistically different (P< 0.001), as judged fiom their peak melting

temperatures. Throughout the analysis at various tirnes, two forms of the P' polyrnorph

were detected, perhaps suggesting that both the P2' and the B 1 ' could be obtained by static

crystallization of refined cocoa butter. The B form in these experiments refers to the

or f o m V polyrnorphic form. We did not observe a solid state species with a peak

melting temperature or XRD pattern characteristic of the P 1 form at any time-temperature

combination.

Some of the polyrnorphic forms detemined fkom peak melting temperatures

obtained by DSC were confumed using powder XRD. The short spacings that we

obtained were identified as a, B', or B by cornparison with published work (Table 7)

(Larsson, 1994). Using DSC, a pure y form was not observed, even at low temperanires.

XRD allowed for the determination of a polyrnorphic form after 2 minutes of

crystallization at a particular temperature. Static crystallization for 2 minutes at -20°C

gave an XRD pattern with short spacings at 3.79A and 4.19A (Figure 10A) characteristic

of the y polymorphic form. These short spacings are also characteristic of the P' form but

i~ this case we know the y polymorph is present since the next polyrnorph to form was

the a form. Polyrnorphic transitions occur only from less stable polymorphs to more

stable forrns and are irreversible.

Table 7: Characteristic short spacings as determined by XRD for the vaxious polymorphic forms of cocoa butter (Larsson, 1994).

Polymorphic Short Spacings (A) form

y (sub-a) 3.87(m), 4.17(s) a 4.20(vs)

Pz' 3-87(vw), 4.20(vs) Pi' 3.75(m), 3.88(w), 4.13(s), 4.32(s) P 2 3.65(s), 3.73(s), 3.87(w), 3.98(s), 4.22(w), 4.58(vs), 5.13(w).

5.3 8(m) Pl 3.67(s), 3.84(m), 4.01(w), 4.21(vw), 4.53(vs), 5.09(vw), 5.37(m)

The relative intensity is noted as very strong (vs), strong (s), medium (m), weak (w) or very weak (vw).

After crystallization for 2 minutes at 5"C, a short spacing was observed fiom the XRD

patterns at 4.2 1 A (Figure 1 OB). This value is characteristic of the a form and confirms

the polymorphic form determined by DSC. M e r 5 days of incubation at 5OC the B' form

was determined by DSC and confirmed using XRD by the observation of short spacings

at 3.8 1 A and 4.1 8A (Figure 1 OC). After crystallization at 22OC for 28 days, the

diffraction pattern displayed short spacings at 3 J O & 3.94A, 4.58A and 5.42A (Figure

10D); characteristic of the B form. Cocoa butter crystallized statically at 2Z°C for 28 days

had a peak melting temperature of 32.i'OCy which is also characteristic of the P

polyrnorph.

Figure 10: Characteristic X-ray diffkaction patterns of the various polymorphic forms of cocoa butter crystallized at -20 OC for 2 minutes (A), 5 O C for 2 minutes (B), 5 OC for 5 days (C) and 22 OC for 28 days @).

From the peak rnelting temperatures, two P' polymorphs were found to exist. However,

this was not confirmed by XRD. Only the pzy fom, with characteristic short spacings at

3.87A and 4.20A was observed (Larsson, 1994). In many instances where the B 1 ' was

detected by DSC, the P polymorph was also present. It appears that in regions where

both of these polymorphic forms are present, the short spacings have values which more

closely resemble those of the P form.

DSC melting profiles were deterrnined for cocoa butter, which was statically

crystallized £rom an 80°C melt to temperatures ranging fiom -20°C to 26°C. Incubation

times ranged anywhere fiom 1 hour to 35 days depending on the crystallization

temperature. Results from these DSC expenments were confirmed by powder XRD.

This data was used to construct a tirne-temperature state diagram for the polymorphism

of statically crystallized cocoa butter (Figure 11).

At crystallization temperatures between -15°C and -20°C, both the y and the a

polymorphs were observed for a 7 day period. Afier 2 minutes at -20°C, the y form was

observed by XRD but not by DSC. This was Iikely due to the limitations of the DSC

equipment, as previously discussed in this section. The melting profiles obtained by DSC

indicated that only the a polymorph was present at -1 5°C and -20°C afier 4 minutes of

crystallization. Subsequently, we detected the existence of the y form concurrently with

the dominant a polyrnorph; both remained stable for 7 days. This does not indicate that

cocoa butter crystallized into a more stable state and then rearranged into a less stable

state. Rather, as previously discussed, we speculated that fractionation had occurred in

which some of the triacylglycerols remained in the more stable a conformation while

others crystallized into the unstable y polymorph.

100 1000

Time (min)

Figure 11: Time-temperature state diagrarn for the polyrnorphism of statically crystallized cocoa butter. Syrnbol(*) represents the polymorphic forms that have been determined by XRD.

At crystallization temperatures ranging fkom -1 0°C to 20°C, the cr polymorph was

initially observed, as determined by DSC. The y polyrnorph has been observed by XRD,

to form initially at crystallization temperatures ffom -10°C to 3°C and rernain stable for 3

minutes (van Mafssen et al., 1999). Once again, we Cid not obtain this result likely due to

the limitations of the DSC equipment. However, the initial formation of the a polyrnorph

at 5°C and 20°C was confirmed by XRD in our study. The a polymorphic form was

found to be quite stable at low crystallization temperatures. At -1 0°C and -5°C the a

pol-yrnorph remained stable even after 7 days of incubation. At crystallization

temperatures higher than -5"C, the a polyrnorph was found to remain stable for

incubation times ranging fkom 10 minutes (1 7.5"C and 20°C) to 2 days (0°C) depending

on the crystallization temperature.

At crystallization temperatures ranging fkom 0°C to 20°C, a polymorphic

transition region fkom the a polymorph to the B' form was observed. At low

crystallization temperatures, the compIete transformation of the less stable a polymorph

to the more stable P' polymorph can require up to 8 days of incubation while at higher

temperatures the transformation requires only 40 minutes.

The state diagram (Figure 1 l), clearly shows that the P' polymorph can be formed

directly fiom the melt or from the melt via the a polyrnorph. At temperatures from 2 1 OC

to 26°C crystallization of the P' polymorph c m take from 1 h o u to 3 days. The stability

of the P' polymorph is dependent on the crystallization temperature. At lS°C, the B'

form remained stable for 28 days, whereas at the higher temperatures, a transformation to

the more stable P form occurred d e r a few hours.

The second polyrnorphic transition region, fiom the P' polymorph to the B form,

took place at crystaIlization temperatures in the range 20°C to 26°C. At 20°C this

transition occurred over a 3 week period, while at 25OC and 26"C, the transition was

complete within hours. Finally, the P form was observed to crystallize fiom the melt

either ~ o m the a form via the B7polyrnorph, or directly fiom the B' polymorph. This

stable polyrnorphic form was only observed at higher crystallization temperatures (20°C

to 26°C) and in some cases only after incubation times of 35 days. The P polymorph was

found to remain stable for several weeks of storage at crystallization temperatures

ranging from 22°C to 26°C.

An anomalous region, termed y SOS, was detected at 25OC and 26°C. After

crystaIIization at 25°C for 20 hours as well as 26°C for 1 day, the first crystal structure

formed displayed a melting range fiom 352°C to 37.3OC. Initially, we thought that this

particular crystal form could be the P 1 polyrnorph. However, after crystallization at 25°C

for 1 day and at 26OC for 3 days, results fiom both DSC and XRD indicated the existence

of the B' form. It is very unlikely that the stable P 1 would transform to the more unstable

p' indicating that the initial crystal form at these temperatures was not the P I polyrnorph.

The XRD pattern of this crystal form displayed a broad short spacing at 4 . 4 ~ 4 (Figure

12). This also confirmed that this particular crystal was not the P l polymorph found in -

cocoa butter. Much work involving the detemination of the various polyrnorphic forms

of POP, POS and SOS, the main triacylglycerols in cocoa butter has been carried out

(Arishima et al., 199 1 ; Sato et al., 1989).

Figure 12: XRD pattern of cocoa butter statically crystallized at 26°C for 1 day.

The y polymorphic form of SOS was found to form directly &om the melt at a

crystallization temperature between 24°C and 28°C (Sato et al., 1989). The y form of

SOS was aiso determined by DSC to have a melting temperature of 35.4"C (Table l), a d

XRD patterns illustrated two sharp short spacings at 3.88A and 4.72A and a broad short

spacing at 4.5A (Sato et al., 1989). In this work, we observed a polymorphic form with a

sirnilar melting point and a broad short spacing at 4.45A. It was hypothesized that the

SOS fraction of cocoa butter crystallized initially, with the rernainder of the

triacylglycerols remaining in the melt. Thereafter, the SOS rearranged and CO-

crystallized with POP, POS, and the rninor triacylglycerols into the B' crystal fom.

van Malssen et al. (1999) had previously constnicted a state diagram for the

polymorphism of statically crystallized cocoa butter. Even though van Malssen et al.'s

(1999) and our state diagram displayed sirnilar &ends, polymorphic transitions occurred

more rapidly in their study. This may be due to the fact that the cocoa butter used by van

Malssen et al. (1999) was not refined. On the other hand, the cocoa butter used in our

experiments was refined and had a decreased phospholipid content. Degumrned cocoa

butter has been found to show a significantly slower crystallization rates relative to

unrefined cocoa butter (Dimick, 1 999). When crude degumrned material, which had a

melting point very close to that of lecithin, and a pure sn-1,2-distearoyl-

phosphatidylcholine were added back to the refined cocoa butter, the nucleation induction

tirne was reduced, and the crystallization growth rate was increased (as reported by

Dimick, 1999). The refined cocoa butter used in our research would therefore be

expected to have lower crystallization rates compared to unrefined cocoa butter.

It m u t be stressed that this state diagram is specific to the cocoa butter used in

this study. This particular cocoa butter has been degummed, deodorized, and bleached,

and therefore is lacking in phospholipids and other minor components, which most likely

slow down its crystallization. In addition, the absence of phospholipids may also slow

down polymorphic transformations.

Crystallization Kinetics

When a fat is in the liquid state, triacylglycerol molecules are in random motion.

Supercooling of the fat causes molecules to cluster and pack together, which eventually

leads to the formation of a crystal nucleus. At high degrees of supercooling, thïs

nucleation process is very rapid. In this instance, molecules have little time to rearrange

and are not able to adopt their most thermodynamically favorable conformation. This

leads to the formation of a less ordered solid. At high degrees of supercooling, however,

a rapid increase in viscosity also takes place, which lirnits heat and mass transfer. The

rearrangement of triacylglycerol molecules into a more stable form is thus hindered. The

rate of nucleation as well as the increased viscosity limits crystal growth, leading to a

system with a large number of small crystals. When liquid fat is subjected to a lower

degree of supercooling, triacylglycerol molecules have a greater tendency to arrange

themselves into a more thermodynamically favourable conformation resulting in a more

ordered system. As well, viscosity does not increase to the same extent and the system

has thus a decreased propensity to become heat and mas-transfer limited. At higher

temperatures, nucleation is slower and crystal growth is more extensive than for systems

at high degrees of supercooling. The particular crystal f o m of a fat system is not

determined solely by chernical composition. Both chernical composition and heat and

mass transfer play an important role in the fmal crystal structure and cannot be

considered independently for fat crystallization. For example, it is difficult to determine

whether an unstable crystal form was created as a result of the unfavourable

conformation of the û-iacylglycerol molecules due to rapid nucleation or whether a rapid

increase in viscosity limits the mass and heat transfer making rearrangements of the

molecules very difficult. Both of these factors are responsible for the resultant crystal

forrn.

Crystallization involves nucleation and growth. The mechanism by which

structure develops is best determined by considering these two steps separately.

However, in most fat systems it is quite difficult to distinguish between nucleation and

growth. The Avrarni model has been used in the study of fat crystallization (Dibildox-

Alvarado and Toro-Vazquez 1997; Herrera and Marques Rocha, 1996; Kawamura, 1979;

Metin and Hatel, 1998; Wright et al., 2000; Ziegleder, 1990). The Avrami model c m be

used to quantifi crystallization kinetics and describes the overall crystallization process

taking into account both nucleation and crystal growth (Avrami, 1939; Avrarni, 1940;

Avrarni, 1941).

The crystallization behaviour of cocoa butter was examined in our work by

measuring the increase in SFC as a function of time by puised Nüclear Magnetic

Resonance @NMR). Crystallization curves for cocoa butter crystallized statically at

-20°C, 10°C, 15°C (Figure 13A), 17S°C, 20°C and 22S°C (Figure 13B) were

detennined.

Time (minutes)

100 ,

0 1000 2000 3000

Tirne (minutes)

Figure 13: Crystallization c w e s of cocoa butter crystallized at -20°C (i), 10°C (V), lS°C (A), (A) and 17.S°C (w), 20°C (V) and 22S°C (A) (B). Symbols represent the average f standard error of three replicates.

These curves are characterized by an initial lag penod followed by a rapid increase in

crystal m a s , which levels off at a particular SFC depending on the crystallization

temperature. A high degree of supercooling (-20°C to 1 SOC) leads to a short lag period

and the SFC increase is dmost instantaneous. These curves tend to Zevel off usually

within the f ~ s r 10 minutes of crystallization at very high SFC values (>90%). At lower

degees of supercooling (20°C to 26"C), the initial lag penod is much longer as is the

subsequent increase in crystal mass. Crystallization curves obtained at 25°C and 26T

may take up to 10 days to level off and only reach an SFC value of 60%.

The crystallization curves were fitted to the Avrarni equation (equation (1)) by

nonlinear regression (Marangoni, 19981, in order to quanti& the crystallization kinetics

and to gain insight into the nature of the crystal growth process. The Avrarni exponent,

n, which is sensitive to both the time dependence of nucleation and the dimensionality of

growth was detennined and plotted as a function of crystallization temperature (Figure

14). Statistically, two different regions were determined from this graph (P<O.001). The

first region was determined at crystallization temperatures ranging fiom -20°C to 15°C

and the second region ranged fiom 20°C to 26OC. No statistical differences were

observed within these two regions (P>0.05). The value of n in the first region was less

than 1 .O whereas in the second region a value of about 3 .O was obtained. n i e Avrami

exponent was constant at low crystallization temperatures, followed by a drastic increase

at 20°C. A gradual increase in n was observed about 20°C with increasing temperature.

Similar results were observed for statically crystallized milkfat (Wright et al., 2000). The

changes in the Avrami exponent at 20°C also correspond to changes in microstructure.

Crystallization Temperature (OC)

Figure 14: Changes in the Avrami exponent as a function of crystallization temperature. Symbols represent the average f standard error of the mean of three replicates.

Polarized light micrographs (Figure 18) show a granuiar rnorphology for crystallization

beIow 20°C. At ternperatures of 20°C and above, both spherulite clusters and needle-like

morphologies were observed. These microstnictures indicate difEerences in the crystal

growth geornetry above and below 20°C. Therefore, the Avrarni exponent could be used

to discern between different mechanisms of crystallization.

At crystallization temperatures ranging fiom -20°C to O0C, the a polyrnorph is

predominant. The 0' polymorph is the main form at crystallization temperatures between

5OC and 22S°C, while the major form at 25OC and 26°C is the B polymorph (Figure 14).

The Avrarni exponent was found to change dramatically at crystallization temperatures

above and below 20°C, suggesting that it is not a good predictor of the predominant

polymorphic fom. However, at low crystallization temperatures, less stable a and B'

polyrnorphs are present whereas at higher crystallization temperatures, the P stable

polymorph is predorninant. The Avrarni exponent cm be used to distinguish between the

unstable polyrnorphic forms (a and P ') and the more stable B polymorph.

The induction time is defrned as the point where the amount of solid fat is

significantly different from zero (Sharples, 1966). Induction times were detennined in

our work fiom the cqstallization cuves by extrapolating fkom the linearly increasing

region back to the time axis (Wright et al., 2000). The induction times were then plotted

as a fünction of crystallization temperature (Figure 15). At low crystallization

temperatures (-20°C to O°C), we observed a positive linear relationship between

induction time and crystallization temperature, which then leveled off frorn 5°C to 1 5°C.

A discontuiuity between crystallization temperatures of 15°C and 17S°C was followed

by a positive linear relationship fiom 1 7S°C to 22S°C. From 22S°C to 25"C, another

discontinuity was observed. An increase was also observed between 25°C and 26°C. Et

has been suggested that discontinuities in the relationship between induction time and

crystallization temperature rnay be associated with different polymorphic forms

(Dibildox-Alvarado and Toro-Vazquez, 1997). For tripalmitin, nucleation rate has been

shown to be inversely related to induction time and a discontinuity was observec! at

temperatures where the B' form transforms to the p form (Kellens, et al., 1992).

Discontinuities found in the relationship between induction time and crystallization

temperature in our work are also associated with polymorphic transformations. At low

crystallization temperatures (-20°C to O°C), cocoa butter crystallizes mainly in the a

form. However, we also observed the CO-existence of the y and the or forms at

crystallization temperatures in the range -20°C to -1 5°C. The a polymorph is initially

present at crystallization temperatures ranging fiom 5OC to 15°C but it converts to the P'

polymorphic fonn after 6 hours at 5OC, and 40 minutes at 15°C.

Crystallization Temperature (OC)

Figure 15: Induction times for statically crystallized cocoa butter as a fûnction of crystallization temperature. The polymorphic designation indicates the dominant polymorphic f o m present at a particular crystallization temperature as deterrnined fiom melting points and XRD patterns. Symbols represent the average f standard error of three replicates.

At crystallization temperatures ranging from 17.5OC to 22.50C7 the major form is the B 7

polymorph. At 25°C and 26"C7 we suspected that the initial crystallization was of the

SOS fraction of cocoa butter followed by the formation of the P' polyrnorph, which

quickly transformed to the P polymorphic fom.

Relationships between crystallization kinetics and polymoïphism were sought by

comparing results obtained by pNMR and DSC at the early stages of crystallization

(Figure 16). At a crystallization ternperature of 1 7S°C, the a polymorph formed after 4

minutes stnd remained stable for 10 minutes, as determined by DSC (Figure 16A). For

the next 30 minutes, a polymorphic transition to the more stable P' form occurred.

Finally, after one hou, the P' polyrnorph was the only crystal form present. A sirnilar

pattern could be observed from the crystallization kinetic data obtained at 17.5"C (Figure

16B). An initial increase in SFC during the first 10 minutes was followed by a leveling

off of the c w e until40 minutes. This was followed by a dramatic increase in the SFC

until60 minutes where the SFC plateaus again. The initial linear increase in the

crystallization curve indicates the formation of the a polyrnorph, while the second

increase is associated with the formation of the B' polymorph. The plateau observed

from 10 to 40 minutes corresponds to the region of a to P' polymorphic transformation,

as detemiined by DSC. Figure 16 demonstrates that, in some cases, rneasuring SFC as a

function of tirne provides a good indication of both the formation and the transformation

of the various polymorphic forms.

Time (minutes) 1 O0 -

B

Time (minutes)

Figure 16: Cornparison of the polymorphic forms as determined fiom peak temperatures obtained fiom DSC melting profiles (A) and from crystallization curves (B) of statically crystallized cocoa butter. Syrnbols represent the average f standard error of three replicates.

Microstructure

The microstructural level has a significant impact on the macroscopic rheological

properties of a fat system (deMan and Beers, 1987; Heertje et al., 1987; Narine and

Marangoni, 1999a). This microstructure is dependent on the molecular composition, as

well as its crystallization behaviour, including polyrnorphism. Crystallization kinetics

and polymorphism are dependent on processing conditions, which in turn influence the

resultant microstructure (Narine and Marangoni, 1999a). In this study, polarized light

microscopy (PLM) was used to investigate the microstructures of the various

polymorphic forms of statically crystallized cocoa butter as deterrnined fiorn the time-

temperature state diagram (Figure 11).

Images of the a polyrnorph of cocoa butter displayed a granular appearance

(Figure 17). We observed the CO-existence of the y polymorph and the a form at -20°C

and -1 5OC. However, y was not the dominant polymorphic form. Although these images

were obtained using different processing conditions, their morphologies were quite

sirnilar. Sirnilar images of the unstable a form were observed by Vaeck (1960) and were

descnbed as a very bright crystalline mass. Polarized light micrographs of the B'

polyrnorph were strikingly different and dependent on processing conditions (Figure 18).

Images of P' polyrnorph obtained via the a form at low crystallization temperatures (O to

10°C) were sirnilar to the a crystal form in that they possessed a granular morphology

(Figure 18A and B).

Figure 17: Images obtaùied by PLM of the alpha form of cocoa butter crystallized at -20 O C for 1 day (A), -20 OC for 7 days (B), -1 5 OC for 7 days (C) , and O O C for 1 day (D).

Figure 18: Micrographs of the P' form obtained by static crystallization at O°C for 14 days (A), 10°C for 5 days (B), 15°C for 14 days (C), 20°C for 1 day @), 22°C for 1 day (E), and 24°C for 3 days (F).

The P' polymorph crystallized via the cc form observed at 15'C also had a

granular texture but there was some evidence that the crystals were beginning to cluster

(Figure 18C). At 20°C, the a form is very unstable and the transition to the 9 '

polymorph is quite rapid. At this crystallization temperature the clustering of spherulites

was very evident (Figure 18D). These clusters of sphedites were found to measure

approxirnately 6 0 p . Others research groups have observed clustering of sphenilites at

20°C (Vaeck, 1960). Incubation of cocoa butter for 1 day at 22°C resulted in a similar

microstructure (Figure 18E) to that observed at 20°C, even though the P' polymorph was

crystallized directly from the melt. At 24°C: the P' polymorph was also forrned directly

fiom the melt, but the crystallites, which measured approxirnately 25w, had a needle-

like appearance (Figure 18F). The microstructure of the P' polyrnorph seems to have a

greater dependence on the crystallization temperature than on the path by which it is

formed.

The P polymorph can also display different microstructures (Figure 19). At

crystallization temperatures of 20°C and 22"C, long incubation times of 4 or 5 weeks

were required in order to obtain a P polymorph. After these extended incubation times,

the morphology witiiin the same sample was no longer unifom at crystallization

temperatures of 20°C and 22°C. A continuous phase was observed with a morphology

similar to that seen in the earlier stages of crystallization. Also, large microstructures

(600 pn to 2mm) were observed at later stages of crystallization. After 4 weeks at 20°C

(Figure 19A) clusters of spherulites were observed as the continuous microstructure but

the clustering was less distinct than that observed early on in the ciystailization process.

Figure 19: Images of the stable P form of cocoa butter statically crystallized at 20°C for 28 days (A), 22°C for 28 days (C and D), and 26°C for 28 days (E and F).

The large microstructures (Figure 19B) had a feather-like appearance and were often

large enough to be seen by the naked eye. A similar structure was observed for cocoa

butter crystallized at 22°C for 28 days (Figure 19C and D). At crystallization

temperatures of 24°C and 26OC, the P poIyrnorph displayed a needle-like appearance

(Figure 19E) and after seven days of incubation a second microstructure was observed.

In this case, large microstructures (200pm to 500pm) with a granular center surrounded

by needle-like crystallites were observed (Figure 19F).

The appearance of two different microstnictures, afier a long incubation period,

was speculated to be a result of phase separation, fiactionation or a combination of both.

A phase separation occurs when a material exhibits different States of matter but the same

molecukx composition, as in the case of polymorphism. Fractionation as defined in the

polymorphism section takes place upon a separation of different molecular species. The

observed differences in microstructure as a fünction of time in our work may have been a

phase separation since they were observed in the P phase.

At al1 of the crystallization temperatures used in this research, the P polymorph

was not found to crystallize directly from the melt. During the phase transition fiom the

B' form to the B polymorph, larger microstructures may have formed. The continuous

microstructure being that of the B' polymorph may have remained stable during the

polymorphic transition resulting in the presence of two different microstructures within

the same sample. Further evidence of phase separation stems fiom the fact that at 20°C

and 22OC the existence of two microstructures was observed after a longer incubation

time than for 24°C and 26°C. The polymorphic transition fiom the P' form to the P

polyrnorph occurs after a few days at 24°C and 26"C, but takes several weeks at 20°C

and 22°C. The existence of two microstructures was not observed until28 days at 22°C

but after 7 days at 24°C and 26°C. Tt is also possible that after a month of incubation at

high temperatures a fiactionation of the various triacylglycerols may have occurred.

Studies conducted to relate morphology and compositional characteristics in cocoa butter

show that the formation of different microstructures over time have different molecular

compositions (Manning and Dimick, 1985). At 26°C both "feather crystals" and

"individual" crystals were obsemed within the sarne sample (Manning and Dimick,

1985). We described these as large microstructures with a granular center surrounded by

needle-like crystallites ("feather crystals") and needle-like crystals individual al"

crystals). Tnacylglycerol analysis of the two microstnictures identified by Manning and

Dimick (1985) indicated that the feather crystals and the individual crystals exhibited

significant increases in SOS and significant decreases in POP wher, compared to the

liquid cocoa butter sample. The triacylglycerol composition of these two microstnictures

was similar perhaps suggesting the occurrence of phase separation. However, this work

did not examine the composition of the minor triacylglycerols (PPP, PPO etc.). A

difference in the composition of these molecular species may also suggest that

fiactionation was responsible for the different microstructures. Due to the complex

behaviour of natural fats such as cocoa butter it is likely that the presence of two

microstructures within the sarne sampIe is a result of both phase separation and

fiactionation.

Al1 of the microstructures observed of the a polymorph of cocoa butter had a

granular appearance. However, the P' polymorph and the f! form c m be associated with

different microstructures depending on the degree of supercooling. Optical micrographs

of tripalmitin also suggest that a wide range of morphologies can exist for one

polyrnorphic form (Kellens et al., 1992). When microscopy is used to study cocoa butter

polymorphism, either melting point determination or XRD patterns should be used to

confirm the results.

So far, this discussion has addressed the appearance of the various polyrnorphic

forms of cocoa butter obtained by crystallization at different temperatures. However,

different polymorphic forms are not only observed at different temperatures but also at

different times at the sarne temperature. The previous discussion of morphology

concentrated on the differences observed when moving verticaily along the time-

temperature state diagram, but differences in polymorphism are also evident when

moving honzontally along the state diagram (Figure 11). At low ternperatures

differences in morphology were not observed even after 28 days of storage. Following

crystallization of cocoa butter at 0°C for I day (Figure 20A), crystals are in the a

polymorph and show a granular morphology. During an incubation of 4 to 10 days, a

polyrnorphic transition occurred from the a form to the more stable B' polyrnorph, and

the morphology observed during this transition was again granular and uniform (Figure

20B and C ) . Finally, the P' form was found to remain stable fiom 10 to 28 days and a

granular morphology was observed although a cornplete polyrnorphic transition had taken

place (Figure 20QE and F). At low crystallization temperatures there is a very high solid

fat content, which greatly limits mass transfer. Once the crystals form a particular

microstructure, rearrangernent is very difficult due to the high viscosity of the medium.

At low crystallization temperatures (0°C to 10°C) the microstmcture did not change with

changes in polymorphism.

Figure 20: PLM images of cocoa butter statically crystallized at O°C for 1 day (A), 5 days @), 7 days (C), 14 days @), 21 days (E), and 28 days (F).

At a crystallization temperature of 15"C, the a polyrnorph is only stable for the

fust 20 minutes so images were not obtained since only 8% of the cocoa butter was solid.

AI1 images shown in Figure 2 1 correspond to the P' polyrnorph obtained via the a form

at 15°C. Frorn 1 day to 28 days, the microstructure was found to have a granula texture

but some dustering of the crystallites was evident (Figure 21A, B, D, E and G). At al1

tirnes after 7 days of storage, two difEerent rnicrostnictures were observed, the continuous

granular rnorphology and large spherulitic microstructures (Figure 21C, F and H). The

large microstructures ( - 1 0 0 ~ to 600pm) were only observed in a few of the samples

incubated at lS°C for 7 days. After 28 days, these large microstructures were present in

ail of the samples dong with the granular microstructure (Figure 21G and H). Since the

p' polymorph was found to remain stable for 28 days at 15°C we speculate that the

presence of two different microstructures within the sample is a result of cocoa butter

fkactionation.

Figure 21: PLM images of cocoa butter crystallizedat 15°C for 1 day (A), 7 days (B and C), 14 days @), 21 days (E and F), and 28 days (G and H).

Static crystallization of cocoa butter at 20°C revealed some changes in

microstructure during 35 days of storage (Figure 22). PLM images of the B' polymorph

formed after 1 day of crystallization at 20°C showed clusters of sphenilites (Figure 22A).

These clusters started growing into each other during incubation (Figure 22B and C),

which coincided with the P ' to P polymorphic transition, as determined fkom the state

diagram. By the end of the polymorphic transition, the individual clusters were difficult

to distinguish due to the substantial increase in size (Figure 22D and F). Also, at this

point extremely large microstructures 'were observed in the same sarnple (Figure 22E and

G). As mentioned earlier we speculated this to be a resuit of phase separation,

fiactionation or a combination of both. After 35 days of incubation the polymorphic

transformation is complete and the crystals are in the P polymorphic form. The two

different microstructures observed at 21 and 28 days were still present and the clusters

had grown into one another and were no longer distinct resulting in a granular

morphology (Figure 22H). The large microstructures have a center with a granular

morphology sirnilar to that of the a polymorph but the individual crystallites are much

larger (Figure 221). On the periphery of this granular center there appears to be a distinct

feather crystai growth (Figure 225). The spherulite clusters are evident throughout the 3 5

day incubation but become less distinct over time. The existence of two microstructures

at 20°C after 2 1 days suggests that a phase separation may have occurred since a

polyrnorphic transition fiom the B' polymorph to the P polymorph occurs at this time.

Figure 22: Polarized Light microscope images of cocoa butter statically crystallized at 20°C for 1 day (A), 5 days (B), 7 days (C), 21 days @ and E), 28 days (F and G) and 35 days (H, 1 and J).

The initial microstructure observed following 1 day of incubation at 26°C was

characterized by small crystallites and some larger needle-like crystals (- 2 5 p ) (Figure

23A). Although we were unsure of the polyrnorphic f o m present under these conditions,

results obtained through DSC and XRD analysis ihplied that this was the y polymorph of

an SOS-rich fraction. The B' polymorph, crystallized at 26°C had crystallites (-50~)

with a needle-like appearance (Figue 23B). The needle-like morphology was obsenred

at al1 stages of the 28 day incubation at 26°C (Figure 23C, E, G). Manning and Dimick

(1985) also observed a needle-like microstructure at 26OC. Three days after the P' to P

polyrnorphic transition, Iarge spherditic microstructures (-1 OOpm to 6OOpm) were

observed (Figure 23D, F and H). Similar to the large microstructures seen at 20°C a

granular center was observed but this time the penphery had a needle-like appearance.

Once again the existence of these two microstructures within the same sampIe may be the

result of phase separation, fiactionation or a combination of both. Very similar

microstnictures were obsewed at 24OC except after 1 day the crystallites had a needle-

like appearance unlike the microstructure observed after 1 day at 26°C.

Figure 23: Images obtained by PLM of cocoa butter crystallized at 26OC for 1 day (A), 3 days (B), 7 days (C and D), 14 days (E and F), 28 days (G and H).

The microstructure of the various polyrnorphic forms is a function of both

temperature and time. Cocoa butter crystallized at different temperatures may have the

same polymorphic form but the microstructure can be quite different. At low

crystallization temperatures there is very little change in the microstructure during

incubation due to heat and mass transfer limitations. At higher incubation temperatures

only one microstructure remains stable over time, but a different microstructure fonns

most likely as a result of phase separation, fiactionation or a combination of both. From

these PLM images we are able to assign a set of particdar microstnictiires to a particular

polyrnorphic form. Until recently, microstructure was only used as a qualitative tool but

a theory has been developed in which the spatial distribution of m a s can be quantified

fiom PLM images.

Most of the research on the physical properties of cocoa butter has been focused

on establishing relationships between lipid composition or polymorphisrn and

macroscopic properties (Manning m d Dimick, 1985; Vaeck, 1960). However, the

microstructure of the fat crystal network is also known to influence its physical

properties. Macroscopic properties of the fat crystal network are believed to depend on

the nature of the microstructures as they form the level of structure closest to the

macroscopic world (Narine and Marangoni, 1999a). The concept of fkactal geornetry has

been used to characterize the structure of the fat crystal network in various fat systems

(Marangoni and Rousseau, 1 996; Narine and Marangoni, 1 999a). Ln our work the fiactal

dimension was determined fiom in situ PLM images by a method developed by Narine

and Marangoni (1999a). This method uses the theory of mass fiactals and the equation:

N = c(R)~ (13)

where N is the number of reflections (as determined fiom PLM) in a cube of side R, c is a

proportionality constant and D is the fiactal dimension.

Fractal dimensions were determined for cocoa butter crystallized at temperatures

ranging fkom -20°C to 26OC incubated fiom 1 day to 35 days. Figure 24 shows the

fiactal dimension as a fùnction of cryslsallization temperature after 7 days of

crystallization. When the fi-actal dimension was determined as a function of

crystallization temperature two statistically different regions (P< 0.00 1) were found at al1

crystallization times. The first region had crystallization temperatures ranging from -

20°C to 15°C and the fiactd dimension was about 2.12. This region was found to be

statistically different fkom crystallization temperatures ranging frorn 20°C to 26°C. This

second region had a fiactal dimension of about 2.28. The fiactal dimension determined at

high crystallization temperatures is very close to the value of 2.3 1 obtained for cocoa

butter stored at room temperature as determined by Narine and Marangori-; (1 999a). It

has been suggested that a higher fkactal dimension indicates a higher order of packing

(Narine and Marangoni, 1999a). Therefore, at lower degrees of supercooling we

observed a more ordered distribution of mass obtained via different crystallization

mechanisms. The fractal nature of the microstructure c m be related to the mechanical

properties of the network, namely the elastic modulus (G') by:

G' = mm

where <P is the particle volume fraction of solid fat, m depends on the fiactal dimension

of the network and h is a constant dependent on particle properties (Narine and

Marangoni, 1999a).

Crystallization Temperature (OC)

Figure 24: Fractal dimension vs. crystallization temperatures of cocoa butter statically crystallized for 7 days. Symbols represent the average k standard error of at least eight replicates.

Therefore, according to this equation fracta1 dimension and G' are inversely related as

long as the volume fiaction of solid fat and the particle properties remain constant. At

lower degrees of supercooling, we determined a higher fractal dimension suggesting a

lower value of G'.

The fiactal dimension at a particular temperature was also determined as a

function of time (Figure 25). There were no statistical differences in the fracta1

dimension over time at any of the crystallization temperatures (PB 0.05). In most cases

the microstxucture at each crystallization temperature did not change drastically either as

a function of time (Figure 20,21,22 and 23). At the higher temperatures we did observe

the existence of two rnicrostnrctures after long periods of incubation (Figure 22 and 23).

At crystallization temperatures of 0°C and above there were significant changes in

polymorphism as a fùnction of time. In this case, the fractal dimension was not sensitive

to changes in polymorphism. It appears that once the initial microstnicture is set there is

very little rearrangement with time even though there are significant changes in

polymorphism.

Since there were no significant changes at each crystallization temperature as a

function of t h e , the fiactal dimensions were averaged over time and plotted as a function

of crystallization temperature (Figure 26). The relationship between the fiactal

dimension and the crystallization temperature is vey sirnilar to that found between the

Awami exponent and crystallization temperature (Figure 14). In both of these figures we

observed two distinct regions above and below 20°C. The Avrarni exponent is sensitive

to the mechanism of growth, whereas the fiactal dimension describes the spatial

distribution of solid mass within the network.

Tirne (days) Time (days)

2.0 ! 1 I 1 I 2.0 ! 1 1 1

O 2 4 6 8 O 1 O 20 30 4 O

Tirne (days) Time (days)

Figure 25: Fractal Dimension as a fùnction of time of cocoa butter statically crystallized at-20°C (A), 5°C (B), 20°C (C), and 26OC (D). Symbols represent the average + standard error of at least seven replicates.

Crystallization Temperature (OC)

Figure 26: The fiactal dimension determined microscopically vs. crystallization temperature. The fractal dimension is an average of al1 the tirne points measured at each crystallization temperature. Syrnbols represent the average + standard error of at least fort . replicates.

These results suggest that at lower degrees of supercooling, the growth mechanism of the

system leads to a more ordered fat crystal network.

Other Processing Conditions

The work discussed in this section was of cocoa butter melted at 80°C for 30

minutes and then placed at a constant temperature for a pre-detennined incubation time.

We thought that it might be interesting to study the ef5ect of "cold tempering" on the

SFC, polyrnorphism, microstructure and rheological properties of statically crystallized

cocoa butter. "Cold tempering" was achieved by melting cocoa butter sarnples at 80°C

for 30 minutes then placing them at either -15°C or 5°C for 2 days. The sarnples fiom

each temperature were then transferred to incubators set to 20°C, 24°C and 26°C and

analyzed at 7 and 28 days. Polymorphism was determined fiom die peak temperature

obtained fiom DSC melting profiles, SFC was detemined by pNMR and PLM was used

to observe the microstructure.

The SFC of the "cold tempered" samples and the directly crystallized samples

(Table 8) were not significantly different (P>0.05).

Table 8: Solid Fat Content (SFC) for "cold temyered" and directly crystallized cocoa butter held at 20°C, 24°C and 26°C for 28 days. Cold SFC (%) Directly Crystallized SFC (%)

Tempered 20°C 78.6 * 0.80" 20°C 82.3 0.70 a - -

24°C 63.0 * 0.40 a 24°C 60.6 0.20 a

26°C 52.0 1.1 a 26°C 59.3 * 3.9 a

"Mean value of three replicates * standard error of the mean.

Also, the peak melting temperatures obtained by "cold tempering" were not statistically

different from those obtained by direct crystallization (P>0.05). These "cold tempered"

sarnples incubated at 20°C, 24OC and 26OC for 28 days and the samples directly

crystallized at 20°C, 24°C and 26°C for 28 days were al1 found be in the B crystal form.

Holding cocoa butter at -1 5°C or 5°C before crystallizing the samples at higher

temperatures does not seem to have an effect on polymorphism. However, these samples

were much too sofi to be analyzed rheologically. The samples at 34°C and 26°C were

partially liquid and at 20°C the samples were brittle and crumbled when removed fiom

the molds. Samples crystallized directly at 20°C and 24°C were solid and, samples

crystallized at 26OC were partially liquid even after 28 days so G' values could not be

obtained.

n i e microstmcture of the "cold tempered" and directly crystatlized cocoa butter

are strikingly different. Incubation at -1 5°C for 2 days and then at 20°C for 28 days gave

a continuous network of large clusters (- 350 p) with a granular center and feather-like

crystds around the perimeter (Figure 27A). Microstructures as large as 2mrn were

obtained by crystallization of cocoa butter at 5°C for 2 days followed by a 28 day

incubation at 20°C (Figure 27B). These large microstructures have a relatively small

granular center (- 450 pm) with a very long network of feather-like crystals extending

outwards. These images are similar to the large microstructures observed by direct

crystallization at 20°C after 28 days (Figure 27C and D) but in the "cold tempered"

sarnples there is no evidence of a secondary microstructure.

Figure 27: Polarized light microscopy images o f cocoa butter crystallized at -1 5°C for 2 days and then held at 20°C (A) and 24OC (E) for 28 days and of cocoa butter crystallized at 5°C for 2 days and then heid at 20°C (B) and 24°C (F) for 28 days. Images of direct crystallization at 20°C (C and D) and 24OC (G and H) are also shown.

Crystallization at - 1 5°C followed by incubation at 24°C gave a continuous

network of large clusters (- 300 pm) with a granular center surrounded by needle-like

crystals (Figure 27E). An initial temperature of 5°C followed by incübation at 24°C gave

very large microstructures with relatively small granular centers (- 200 prn) surrounded

by many feather-iike crystals (Figure 27F). Once again these microstructures were very

large (2mm) and could be observed without the aid of the microscope. The

microstructures observed after direct crystallization at 24°C gave two microstmctures;

one with a needle-like appearance (Figure 27G) and the other contained large

microstructures with a grandar center surrounded b y needle-like crystallites (Figure

27H).

The microstructural level of the "cold tempered" samples was quantified using the

microscopie fiactal anaiysis. The fkactal dimensions of the samples crystallized initially

at - 1 SOC and placed at 20°C, 24°C and 26°C were found to be statistically different than

those crystallized directly at 20°C, 24°C and 26OC (P<0.001). Extremely large

microstructures were obtained by an initial crystallization at 5OC followed by an

incubation time of 28 days (Figure 278 and F). The fiactal dimension of these samples

was not determined due to the size of the microstructures. Even using a 4X

magnification only part of a single crystd could be observed. This not only makes

thresholding very difficult but to quanti@ the spatial distribution of mass when only part

of a crystal can be observed is not representative of the organization of the entire fat

network. The "cold ternpered" samples held at 20°C for 28 days had a fiactal dimension

of 2.35 as compared to a value of 2-28 determined for cocoa butter directly crystallized at

20°C (P<O.001). Cold tempered sarnples held at 24OC for 28 days had a fiactal

dimension of 2-34, which is statisticdly different from a fiactal dimension of 2.27

observed by direct crystallization (P<0.001). After a 28 day incubation, direct

crystallization at 26°C gave a fiactal dimension of 2-27 whereas a significantly higher

value of 2.35 was determined for the "cold tempered" sarnples held at 26°C (PcO.001).

The fractal dimension values of the "cold tempered" samples were significantly higher

suggesting a more ordered distribution and a Iower G' value.

The elastic properties of the network have been stated to be more dependent on

spatial distribution of solid mass within the network than the total arnount of solid in the

network (Narine and Marangoni, 1999a). This provides a possible explmation as to why

samples with a similar SFC and the same polymorphic form have different hardness

values. In previous work it has been established that the fiactal dimension can be

manipulated by changing the processing conditions (Narine and Marangoni,. 1999a). The

results that we obtained by "cold tempering" compared to direct crystallization confïrms

this finding. Since the rheological properties of the "cold tempered" samples could not

be determined, we were unable to compare the elastic modulus (Gy) with the directly

crystallized samples. However, a higher fiactal dimension has been shown to result in a

lower value of Gy as long as the other parameters including lipid composition, SFC and

polyrnorphism remain constant (Narine and Marangoni, 1999a). This suggests that the

"cold tempered" sarnples would have a lower elastic modulus when compared to the

directly crystallized samples.

CONCLUSIONS

The aim of this work was to establish a relationship between polyrnorphism,

crystallization kinetics, microstnicture and rheological properties of staticdly crystallized

cocoa butter. Induction times and Avrarni exponents were used to qiianti& the

crystallization kinetics of this fat. In this stiidy, the fiactal dimension, a mathematical

indicator of structure, was used to quanti@ the microstructure. Correlation coefficients

were determined for these three parameters (Table 9).

Table 9: The relationship between the n and T, D and z, as well as D and n for statically crystallized cocoa butter.

n and T D and T D a n d n R~ 0.48 0.50 0.95

A weak (r2 = 0.48), but significant, correlation was determined between the induction

time and the Avrami exponent (P = 0.0126). The induction time is related to

polyrnorphism whereas the Avrami exponent seems to depend on a more macroscopic

mode of growth. No significant correlation (r2 = 0.50) was found between the induction

time and fiactal dimension (P = 0.0502) in this work. Induction times and peak melting

temperatures describe changes in structure at the molecular level. Even though the

molecular level of struchire, including lipid composition and polymorphism, is important,

it does not appear to be a good indicator of network microstructure.

On the other hand, a strong correlation (r2 = 0.95) was found between the Avrami

exponent and the fiactal dimension (P<0.00 1). Distinctly different regions above and

below 20°C were evident in both n and D versus crystallization temperature. Also, the

observed microstructure in these two regions was very different. The Awami exponents,

microstructure and fiactal dimensions for these two regions suggest different

macroscopic network growth mechanisms. Moreover, the macroscopic rheological

properties of a fat network have been obsewed to be closely related to its inicrostructural

characteristics. The strong correhtion between D and n is not surprising since both

parameters describe events at the microstmctural, rather than the molecular levels. As

well, the elastic modulus of a fat crystal network is an indicator of the macroscopic

consistency of the fat network (Narine and Marangoni, 1999e). This suggests that in our

work, the Avrami exponent and the fiactal dimension may be used, in some cases, to

predict the mechanicd strength, and possibly the hardness of a fat crystal network.

SUGGESTIONS FOR FUTURE RESEARCH

The next step in this project should be the determination of the elastic moddus

(G') fiom rheologicai methods. This will enable the relationship between fracta!

dimension and G' to be deterrnined. As well, the Gy and D values c m be used to

calculate the parameter y, which is related to particle properties. This may provide

insight into the effect of molecular composition and polyrnorphism on the macroscopic

properties of the fat network.

Another possible direction that this project may take is the cornparison of refined

and unrefined cocoa butter. This thesis describes the relationship between crystallization

behaviour and microstructure of refined cocoa butter. However, it would be valuable to

determine whether minor components such as phospholipids Hected polyrnorphism,

polymorphic transformations, microstructure and rheological properties. It has already

been suggested that phopholipids play an important role in nucleation and their absence

may affect the crystallization mechanism and, in turn, microstructural and mechanical

properties.

When chocolate is manufactured, the cocoa butter along with the other ingredients

are heated and stirred at 4S°C. This liquid chocolate is then cooled down in a tempering

machine so that crystals c m start to form. However, chocolate is a very poor conductor

of heat so for it to cool quickly it must be well mixed or sheared. In many tempering

machines the shear rate is between 3000 and 8000 s-l. hstead of statically crystallizing

cocoa butter, it codd be subjected to processing conditions that more closely resemble

those of chocolate manufacturing. Then a study somewhat similar to that presented in

this thesis codd be carried out. This would allow for a better understanding of the

relationship between crystallization behaviour, microstructure and macroscopic

properties of chocolate.

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