Faculteit Bio-ingenieurswetenschappen

Academiejaar 2010–2011

Structure development in confectionery products: importance of triacylglycerol

composition

Nathalie De Cock Promotor: Prof. dr. ir. Koen De Wettinck Tutor: Dr. Ir. Veerle De Graef

Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen: levensmiddelenwetenschappen en voeding

Faculteit Bio-ingenieurswetenschappen

Academiejaar 2010–2011

Structure development in confectionery products: importance of triacylglycerol

composition

Nathalie De Cock Promotor: Prof. dr. ir. Koen De Wettinck Tutor: Dr. Ir. Veerle De Graef

Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen: levensmiddelenwetenschappen en voeding

Woord vooraf Ik wil graag iedereen bedanken die betrokken was tot het stand komen van mijn thesis. Het was wel

niet altijd eenvoudig uit te leggen waar mijn thesis nu net over gaat, daarom iedereen bedankt om de

moeite te nemen om lang genoeg te luisteren. Het bestuderen van verschillende soorten vetten heeft

geleid tot een nieuw besef van wat een kennis er schuilt achter de structuur van een koekje, een cake

of taart, dat ik graag met iedereen wil delen.

Daarom wil graag eerst mijn promotor Prof. dr. ir. Koen Dewettinck bedanken om me te de kans

geven om bij de vakgroep voedselveiligheid en voedselkwaliteit mijn thesis te doen. Het tot stand

brengen van een thesis was een leerrijke ervaring.

Mijn dank gaat uiteraard ook uit naar mijn tutor Dr. ir. Veerle De Graef. Het hele jaar stond haar deur

voor mij open, geen enkele vraag was te veel. Bedankt om me te begeleiden, van nuttige tips te

voorzien, voor het keer op keer nalezen van mijn schrijfsels en voorzien van oppeppende woorden

wanneer ik het even niet meer zag zitten.

Ook wil ik graag alle andere personeelsleden van de vakgroep bedanken voor de toffe sfeer, voor het

helpen in nood en voor het verdragen van mijn af en toe klungelige gedrag. Dan denk ik bijvoorbeeld

aan Nathalie De Clerq voor het steeds weer verhelderen van de reservatielijst, Het hebben van

dezelfde voornaam en initialen kan duidelijk voor heel wat verwarring zorgen. Of aan Benny voor het

oplossen van de vaak zich zelf uitwijzende technische problemen en de toffe mopjes. En aan Bea voor

het met veel geduld uitleggen van het integreren van GC pieken.

Daarnaast wil ik graag ook alle thesisstudenten bedanken waarmee ik dit jaar het labo heb gedeeld

voor de leuke sfeer en de toffe babbels. Dank je wel Liesbeth, Elien, Thomas, Kwinten en Dieter.

Thomas bedankt voor het veelvuldig insteken van mijn reologie stalen. Elien en Liesbet voor het

helpen wanneer ik weer eens vergeten was hoe ik nu weer de NMR moest opstarten.

Ook ik wil ik nog Sabine Danthine, van de universiteit van Liège, Gembloux Agro Bio Tech, Unité de

Valorisation des bioressources, Laboratoire de Science des Aliments et Formulation bedanken voor

het uitvoeren van de XRD analyse.

Vervolgens wil ik Loders Crocklaan (Wormeveer, the Netherlands) bedanken voor het voorzien van

de startoliën en voor het uitvoeren van de HPLC analyse op die startoliën. Daarbij wil ik ook graag

Kevin Smith bedanken voor het snel oplossen van de problemen rond de HPLC analyse.

Als laatste wil ik graag familie en vrienden bedanken. Ik wil zeer graag mijn ouders bedanken om

altijd voor me klaar te staan, niet alleen dit jaar maar al heel mijn leven. Niets is jullie te veel. Dank je

wel mama voor het steeds aanhoren van mijn gezaag en me steeds weer op te peppen gedurende mijn

hele academische carrière. Papa voor verhelpen van technische problemen en me weer tot orde roepen

wanneer ik weer eens iets te veel van mijn werk af dwaalde (ook al hoorde ik dit niet altijd even

graag). En natuurlijk mijn broer Laurenz om ondanks jouw eigen studiezorgen, toch maar steeds naar

de mijne te luisteren en voor de vele toffe ontspanningsactiviteiten. Ik zie jullie allemaal doodgraag.

En al mijn vrienden voor doen verdwijnen van de stress met een leuke activiteit, die 5 jaar waren

ongelofelijk leuk.

Nathalie De Cock,

28 mei 2011

i

Contents 1. List of abbreviations ................................................................................................................ 1

2. Abstract ................................................................................................................................... 3

3. Samenvatting ........................................................................................................................... 4

4. Introduction ............................................................................................................................. 5

5. Literature review ..................................................................................................................... 6

5.1 Crystallization ............................................................................................................................... 6

5.1.1 The importance of fat crystallization in commercial products ............................................... 6

5.2.2 Primary crystallization............................................................................................................ 7

5.2.2.1 Driving force ................................................................................................................... 7

5.2.2.2 Nucleation ....................................................................................................................... 7

5.2.3 Crystal growth ........................................................................................................................ 8

5.2.4 Polymorphism ........................................................................................................................ 8

5.2.4.1 Basic polymorphs ............................................................................................................ 8

5.2.4.2 Phase transitions .............................................................................................................. 9

5.2.4.3 Polymorphic behavior of the studied triacylglycerols ................................................... 10

Monoacid TAG such as PPP and SSS ................................................................................... 10

Mixed-acid saturated/unsaturated TAG such as POP and SOS, PPO and SSO .................... 10

5.1.5 Compound crystals ............................................................................................................... 11

5.1.6 Microstructure of fat crystal networks.................................................................................. 11

5.1.7 Mathematical models describing fat crystal networks ......................................................... 13

5.2 Fat modification techniques ........................................................................................................ 13

5.2.1 Hydrogenation ...................................................................................................................... 13

5.2.2 Interesterification .................................................................................................................. 13

5.2.3 Fractionation ......................................................................................................................... 13

5.2.4 Blending ............................................................................................................................... 14

5.3 Phase behavior ............................................................................................................................. 14

5.3.1 Phase diagrams ..................................................................................................................... 14

5.3.2 Phase behavior of PPP/POP ................................................................................................. 16

5.3.3 Phase diagram of POP/PPO.................................................................................................. 16

5.3.4 Phase behavior of SOS/SSO ................................................................................................. 17

5.4 Storage ......................................................................................................................................... 18

5.5 Thesis topic.................................................................................................................................. 20

6. Materials and methods ........................................................................................................... 21

6.1 Substrate ...................................................................................................................................... 21

ii

6.2 Fatty acid and triacylglycerol composition ................................................................................. 21

6.3 Making of the blends ................................................................................................................... 21

6.4 Methods to study fat crystallization ............................................................................................ 21

6.4.1 DSC ...................................................................................................................................... 21

6.4.1.1 Isothermal DSC ............................................................................................................. 22

6.4.1.2 Stop and return DSC ...................................................................................................... 22

6.4.2 Solid Fat Content (SFC) ....................................................................................................... 22

6.4.3 Rheology .............................................................................................................................. 22

6.4.4 XRD...................................................................................................................................... 22

6.4.5 Fractal dimension ................................................................................................................. 23

6.5. Methods to study the storage stability ........................................................................................ 23

6.5.1 Microscopic analysis ............................................................................................................ 23

6.5.2 Hardness measurements ....................................................................................................... 24

6.5.3 SFC measurements ............................................................................................................... 24

7. Results and discussion ........................................................................................................... 25

7.1 Composition of the starting oils .................................................................................................. 25

7.2 General scheme of the research ................................................................................................... 26

7.3 Fat blending ................................................................................................................................. 27

7.4.1 Fatty acid composition of the blends .................................................................................... 27

7.4.2 TAG composition of the blends ........................................................................................... 28

7.5 Crystallization behavior .............................................................................................................. 30

7.5.1 Solid fat content (SFC) analysis ........................................................................................... 30

7.5.2 DSC analysis ........................................................................................................................ 31

7.5.2.1 Isothermal DSC ............................................................................................................. 31

7.5.2.2 stop and return DSC ...................................................................................................... 33

7.5.3 XRD measurements: short spacings (WAXD) ..................................................................... 39

7.5.5 fractal dimension .................................................................................................................. 42

7.5.5 Rheology .............................................................................................................................. 43

7.6 Storage experiments .................................................................................................................... 45

7.6.1 Effect of crystallization temperature .................................................................................... 45

7.6.2 Effect of storage conditions .................................................................................................. 48

7.6.2.1 Evolution of the hardness during storage ...................................................................... 48

7.6.2.2 Evolution of the SFC during storage ............................................................................. 51

7.6.2.3 Evolution of the microstructure during storage ............................................................. 55

Storage at 20°C ...................................................................................................................... 55

Storage at 25°C ...................................................................................................................... 56

iii

Storage at 30°C ...................................................................................................................... 56

Conclusion ............................................................................................................................. 58

8. Conclusion ............................................................................................................................. 59

9. Further research ..................................................................................................................... 61

10. References ......................................................................................................................... 62

11. Appendices ........................................................................................................................ 65

List of abbreviations

1

1. List of abbreviations C14:0 myristic acid

C16:0 palmitic acid (P)

C18:0 stearic acid (S)

C18:1 oleic acid (O)

C18:2 ω-6 linoleic acid (L)

C18:3 ω-3 α-Linolenic acid

DSC Differential Scanning Calorimetry

D fractal dimension

dr distance between the reflecting entities

d euclidic dimension

δ phase angle

fhSHf fully hardend Shea olein

GC Gas Chromatography

|G*| complex modulus

HOSF High Oleic Sunflower Oil

HPLC High Performance Liquid Chromatography

iISHs Interesterified Shea stearin

IPs Interesterified Palm stearin

MUFA Mono Unsaturated Fatty Acids

OOO Oleic Oleic Oleic TAG

PLM Polarized Light Microscopy

PLP Palmitic Linoleic Palmitic TAG

PMF Palm Mid Fraction

pNMR pulsed Nuclear Magnetic Resonance

POP Palmitic Oleic Palmitic TAG

PPO Palmitic Palmitic Oleic TAG

PPP Palmitic Palmitic Palmitic TAG

List of abbreviations

2

Ps Palm stearin

PUFA Poly Unsaturated Fatty Acids

SAFA Saturated Fatty Acids

Sat Saturated

SAXS Small-Angle X-ray Diffraction

SFC Solid Fat Content

SHs Shea stearin

SLS Stearic Linoleic Stearic TAG

SOS Stearic Oleic Stearic TAG

SSO Stearic Stearic Oleic TAG

SSS Stearic Stearic Stearic TAG

TAG triacylglycerols

XRD X-Ray Diffraction

WAXD Wide-Angle X-ray Diffraction

Abstract

3

2. Abstract The aim of this thesis is to study the effect of the ratio symmetric to asymmetric TAG on the

crystallization properties and storage stability of fat blends. To this end, eleven blends with varying

ratios of symmetric/asymmetric TAG, but with an equal amount of saturated fatty acids (40%) are

constructed. Eight of these blends were palmitic based and made by mixing different fractions of palm

oil, the other three were stearic based and made by mixing different fractions of shea butter,

symmetric/asymmetric TAG ratio decreased from blend P1 to P8 and from S1 to S3 These blends

were crystallized at 15°C and 20°C and stored at 20°C, 25°C and 30°C. The effect of chain length is

also taken into consideration by comparing palmitic based blends to stearic based blends. The research

consisted of three parts: part one focused on the composition of starting oils and blends, in part two the

crystallization behavior of the blends was investigated while the storage stability was studied in part

three.

The starting oils and blends were characterized in terms of fatty acid profile (with GC) and TAG

composition (with HPLC). Based on the results of the starting oils eleven blends were constructed that

differed in the ratio symmetric/asymmetric TAG. The crystallization behavior was investigated using

pNMR, DSC (isothermal and stop and return), XRD, fractal dimension determination and rheology.

The DSC results showed a two step crystallization for all blends. The stop and return DSC data

showed that for a crystallization temperature of 15°C the speed of crystallization decreased with the

decreasing symmetric/asymmetric TAG ratio. Furthermore, stop and return DSC data gave an

indication that polymorphic transitions were present in most of the blends. This was confirmed by X-

ray analysis. WAXD data demonstrated an α to β’ polymorphic transition for all palmitic based blends

and for the stearic based blends polymorphic transition happens to β’ or β. Determination of the fractal

dimension showed a linear relation between ln(hardness) and ln(SFC) for both stearic and palmitic

based blends. From both the rheological and the DSC data it was observed that quite some

crystallization took place during cooling as a consequence of its rapid crystallization. The storage

stability of the blends was examined for SFC, hardness and microstructure. At a storage temperature

of 20°C post-hardening occurred for all blends, while at higher storage temperatures (25°C and 30°C)

the hardness decreased upon storage as a consequence of softening of the network and dissolving

crystals. For the stearic based blends a clear effect of symmetric/asymmetric TAG ratio was observed,

the hardness was higher at all storage times and temperatures for those that contained SSO. The SFC

data showed for the stearic based blends that the SFC of blend S2 was always highest during storage,

suggesting that the crystals are most stable when a 3:1 symmetric/asymmetric TAG ratio is chosen

(compound crystals). The microstructure analysis showed a different microstructure for blend S1

compared to S2 and S3 that was characterized by the formation of big crystals imbedded in a network

of small crystals upon storage.

When taking into account all the different analysis it can be concluded that the palmitic based blends

have a similar crystallization behavior, but differ in speed of crystallization, amount of crystallization.

The storage stability and the hardness, SFC and microstructure during storage were similar. For the

stearic based blends the crystallization behavior differed between S1 and S2/S3, S1 crystallized much

faster. The storage experiments showed that S2 was the most stable and that S1 shows development of

large crystals The effect of larger chain length resulted in a much faster crystallization (more

crystallization during cooling) and upon storage a higher SFC, a higher hardness, smaller crystals and

a denser crystal network.

Samenvatting

4

3. Samenvatting Het doel van deze thesis is om het effect van de ratio symmetrische tegenover asymmetrische TAG op

de kristallisatie eigenschappen en de stabiliteit tijdens bewaring van de blends te onderzoeken.

Daarom werden elf blends gemaakt met verschillende symmetrische/asymmetrische TAG ratio maar

met eenzelfde hoeveelheid verzadigde vetzuren (40%) gemaakt. Acht van deze blends zijn palmitine

gebaseerd en zijn gemaakt door het blenden van verschillende fracties van palm olie, de andere drie

zijn stearine gebaseerd en zijn gemaakt door verschillende fracties van shea boter te blenden, de

symmetrische/ asymmetrische TAG ratio daalt van blend P1 naar P8 en van S1 naar S3. Dit onderzoek

valt uiteen in drie delen: de studie van de samenstelling van de startoliën en van de verschillende

blends, de studie van het kristallisatiegedrag en dat van het gedrag tijdens bewaring van de blends.

De samenstelling van de start oliën en blends werd onderzocht via GC voor het bepaling van de

vetzuursamenstelling en HPLC voor het bepalen van de TAG samenstelling. Gebaseerd op de

resultaten voor de start oliën werden elf blends gemaakt met verschillende symmetrische/

assymetrische TAG ratio. Het kristallisatiegedrag werd onderzocht met pNMR, DSC (isotherm and

stop and return), XRD, bepaling van de fractale dimensie en reologie. De resultaten bekomen via DSC

wezen op een 2 staps kristallisatie voor alle blends. Via stop and return DSC werd aangetoond dat de

snelheid van kristallisatie daalde met de dalende symmetrische/asymmetrische TAG ratio bij een

kristallisatie temperatuur van 15°C. Ook gaf de stop and return data een indicatie dat een polymorfe

transitie plaatsvond voor de meeste blends. XRD data toonde een polymorfe transitie van α naar β’

voor alle palmitine gebaseerde blends en voor de stearine gebaseerde blends gebeurde de polymorfe

transitie ofwel naar β’ of naar β. Bij het bepalen van de fractale dimensie is er voor zowel de palmitine

als de stearine gebaseerde blends een lineaire relatie bekomen tussen de ln van de hardheid en de ln

van de SFC. Zowel de data verkregen met DSC als met reologie tonen aan dat voor de stearine

gebaseerde blends nogal wat kristallisatie heeft plaats gevonden tijdens koeling als gevolg van de

snelle kristallisatie. De stabiliteit tijdens bewaring werd geëvalueerd door het meten van de SFC, het

bepalen van de hardheid en het analyseren van de microstructuur. Bewaring bij een temperatuur van

20°C toonde verharding voor alle blends, terwijl bij een hogere temperatuur van bewaring (25°C en

30°C) de hardheid daalde door zachter worden van het netwerk en het oplossen van kristallen. Voor de

stearine gebaseerde blends werd een duidelijke invloed van het verschil in

symmetrische/asymmetrische TAG ratio geobserveerd, de hardheid was bij alle temperaturen van

bewaring hoger voor de blends die meer SSO bevatten. De SFC data toonde dat bij de stearine

gebaseerde blends de SFC altijd het hoogst was voor S2 met een symmetrische/assymetrische TAG

ratio van 3, dus voor dit blend zijn de kristallen het meest stabiel (gemengde kristallen). De

microstructuur analyse toonde een vreemde microstructuur tijdens bewaring voor blend S1: binnen een

netwerk met kleine kristallen waren er grote kristallen aanwezig.

Wanneer er rekening gehouden wordt met alle analyses kan er besloten worden dat de palmitine

gebaseerde blends een gelijkaardig kristallisatie gedrag vertonen, maar verschillen in de snelheid van

kristallisatie en de hoeveelheid kristallisatie. De stabiliteit, de hardheid, de SFC en microstructuur

tijdens bewaring waren gelijkaardig voor de palmitine gebaseerde blends. Voor de stearine gebaseerde

blends was het kristallisatie gedrag van S1 afwijkend van dat van S2 en S3, S1 kristalliseerde veel

sneller. Tijdens bewaring toonde S2 de hoogste stabiliteit en S1 ontwikkelde grote kristallen. Het

effect van een langere keten resulteerde in een snellere kristallisatie (meer kristallisatie tijdens

koeling)en gedurende de bewaring in een hogere SFC,een hogere hardheid, kleinere kristallen en een

denser kristal netwerk.

Introduction

5

4. Introduction Fats form the main structural component in many food products such as for instance butter and

chocolate. Fats consist of a mixture of different TAG and some minor components. The physical

properties of each consisting TAG and the phase behavior of the mixture of these TAG influences the

melting and crystallization behavior, polymorphic transition, crystal morphology and aggregation of a

fat. The crystallization of these TAG forms the basis for the crystal network development of a fat. This

network is directly related to the macroscopic properties of end products such as mouth feel,

spreadability and hardness. Vereecken et al. [29] demonstrated the importance of structural hierarchy

in fat crystallization, with the chemical composition of the fats being an important factor for the final

structure development.

A wide range of modification techniques are used to combine properties of different fats in order to

create a new product with specific physiochemical and technological properties Nowadays, fat

modification is focused on creating more healthy fats that contain less trans and less saturated fatty

acids. This has an enormous impact on the crystallization and polymorphic properties of fats. The

most simple and straightforward fat modification technique is blending of natural fats and oils and is

therefore frequently used. Two types of studies can be discriminated: applied studies and fundamental

studies. Fundamental studies usually investigate mixtures of pure TAG and are consequently too far

away from industrial applications. Applied studies on the other hand focus on the physical behavior of

natural oils and fats and their industrial applicability. The experimental set up of this thesis research

these two approaches are combined as blends of natural fats and oils are investigated from a TAG

point of view.

The objective of this thesis was to study the effect of a different symmetric/asymmetric TAG ratio. So

eleven blends were made by blending different fraction of palm oil for the preparation of the first eight

blends, different fractions of shea butter were blended for the preparation of the 3 last, the symmetric/

asymmetric TAG ratio decreased from P1 to P8 and from S1 to S3. The research consisted of three

parts: the study of the composition of starting oils and blends, that of the crystallization behavior and

that of the storage behavior. The composition was investigated via GC to determine the fatty acid

profile and via HPLC for the TAG composition. The effect of symmetric/asymmetric TAG ratio, chain

length and crystallization temperature on the crystallization is investigated using pNMR, DSC

(isothermal and stop an return), XRD, fractal dimension determination and rheology. The storage

stability of the blends was examined with SFC determination, hardness measurements and

microstructure analysis. For the evaluation of the storage stability the blends were stored at three

different temperatures (20°C, 25° and 30°C) and at two the SFC and hardness or three the

microstructure points in time was evaluated. Next to effect of symmetric/asymmetric ratio,

crystallization temperature, the effect of storage temperature and time is also investigated.

Literature review

6

5. Literature review Fats and oils are used on a major scale in food and other consumer products such as cosmetics,

pharmaceuticals, etc. When focusing on food products, it should be noted that the properties of lipids

are extremely important in products that contain significant amount of fats. Examples of such are

chocolate, confectionary products, dairy products for instance butter and cream, margarine,

shortenings and spreads. Their sensory attributes such as mouth feel, texture, spreadability, the snap in

chocolate are highly influenced by the underlying fat crystal network. Furthermore fats play a

dominant role in the overall structure of the end products. The latter will be discussed in section 1.2.1.

[1, 2, 3, 4].

5.1 Crystallization

5.1.1 The importance of fat crystallization in commercial products

The microstructure and physical properties of commercial products such as margarine or chocolate are

greatly influenced by the behavior of fats. The latter are for the major part present in the crystallized

form. Numerous factors influence the formation of the fat crystal network as is illustrated in Figure 1

[1, 3, 5].

Figure 1: factors influencing the macroscopic properties of a fat crystal network [1], [3]

The chemical composition (TAG and minor components) of a fat and the processing conditions

(temperature, time and shear) affects the primary crystallization behavior of a fat. The latter refers to

when and to what extend a fat crystallizes, polymorphism, polymorphic transitions and crystal

morphology. The TAG crystallize from the melt into distinct polymorphic forms; this will be

discussed in section 5.2.4. The initial size distribution of the crystals is determined by the relative rates

Literature review

7

of nucleation and growth. As the solid fraction increases the individual crystals start touching each

other and aggregate due to Van der Waals forces, resulting in to a slower crystal growth. This

aggregation can already occur at low volume fraction of crystallized fat, leading to voluminous

aggregates while nucleation and crystal growth still continue. Consequently a continuous network is

already formed at low fractions of crystallized fat while a substantial amount of fat still needs to

crystallize. This leads to the formation of solid bridges between crystals and aggregates, generally

referred to as sintering [5].

During storage several post crystallization processes can occur, which affect properties such as

hardness. Polymorphic transition towards more stable phases and changes in the size via Ostwald

ripening can take place. All of the above events don’t necessarily happen chronologically. In addition,

after primary nucleation and successive growth secondary nucleation can take place due to the

presence of growing crystals and can occur together with crystal growth and Ostwald ripening [5].

5.2.2 Primary crystallization

Crystallization can be defined as the first order transition of molecules from a liquid state to a solid

state in a way that the molecules are packed in a regular repeating manner in the solid state. The nature

of the latter depends on the type of bond that is present. In crystals of TAG the bonding is due to Van

der Waals forces and therefore the crystalline state is characterized by closely packed and weakly

attracting TAG [4, 5, 6].

Crystallization comprises several steps. First a sufficient driving force should be provided. Once this is

achieved nucleation occurs, whereby crystal are formed by bringing growth units together to create a

crystal lattice d. From then on crystal growth takes place. Although it is convenient to treat these two

as consecutive events nucleation and crystal growth occur simultaneously [4, 5, 6].

5.2.2.1 Driving force

The driving force for crystallization is the difference in chemical potential between the liquid and solid

phase. The larger this difference, the larger the driving force for crystallization. For crystallization

from the melt the thermodynamic driving force is proportional to the difference in temperature

between the crystallizing system and the melting point. And so the difference in chemical potential can

be written as

(1)

With ∆Hm the molar enthalpy variation of the system during crystallization [J/mol], TKm the absolute

melting temperature [K] and ∆T the supercooling [K] [4, 5, 6].

5.2.2.2 Nucleation

Three types of nucleation are described: primary homogeneous nucleation, primary heterogeneous

nucleation and secondary nucleation. When the nucleation is not catalyzed by the presence of fat

crystals or foreign solid surfaces, it is called primary homogeneous nucleation. For the latter a

supercooling up to 30 K is needed before crystallization can occur. If nucleation is catalyzed by the

presence of foreign surfaces such as dust, container walls; molecules of different compounds, it is

called primary heterogeneous nucleation. Then only a slight supercooling is necessary (1 to 3 K). Most

natural fats contain impurities so that heterogeneous nucleation can occur. Secondary nucleation takes

place when crystals of the crystallizing material are present, and therefore it can only appear after

primary homogeneous or heterogeneous nucleation and subsequent growth. There are three types of

secondary nucleation: apparent, true and contact. The first occurs when crystal fragments of growing

Literature review

8

crystals acts as new nuclei. True secondary nucleation means that crystals are formed in the vicinity of

a crystal of the same phase and not on its surface. Contact secondary nucleation results from collision

of crystals with other crystals, with the walls of the containing vessel or with the impeller [4, 5, 6].

5.2.3 Crystal growth

When nuclei are formed and exceed the crystal size, these become crystallites. Their growth depends

not only on external factors (supersaturation, solvent, temperature, impurities) but also on internal

factors (structure, bonds, defects), so the crystal growth rate can vary by several orders of magnitude.

The actual growth occurs by attachment of molecules to a crystal surface, on the other hand molecules

will also detach. In this way there is a continuous movement of molecules across the surface of the

crystal in both directions. The overall result of these processes determines the growth rate. The

mechanism of how a crystal surface grows depends on the nature of the interface between the crystal

and the liquid. This interface can either be kinked (K), stepped (S) or flat (F). This is illustrated in

Figure 2 [4].

Figure 2: Schematic representation of three types of growth sites. Each cube represents a growth unit [4]

5.2.4 Polymorphism

Polymorphism is defined as the existence of several crystalline phases with the same chemical

composition that have a different structure, but yield identical liquid phases upon melting. Two types

of polymorphism can be distinguished in lipids and organic compounds. When each polymorphic form

is thermodynamically stable in a particular range of temperature and pressure, it is called enantiotropic

polymorphism. Either of the modifications may be the stable one and transformation can go in either

direction, depending on temperature. In monotropic polymorphism one polymorphic form is always

the most thermodynamically stable. Transition from the less stable to the more stable polymorphs will

take place when given sufficient time. Natural fats are invariably monotropic [1, 4, 5].

5.2.4.1 Basic polymorphs

The main structural factor used to characterize the different polymorphs is the subcell structure, this

refers to the packing mode of the hydrocarbon chains of the TAG and the layered structure, which is a

result of the repetitive sequence of the acyl chains which form a unit lamella along the hydrocarbon

axis. A subcell is the smallest spatial unit of repetition along the chain axis. The subcell structure gives

rise to short spacings in X-ray diffraction, whereas the layered gives rise to long spacings. The latter

are dependent on the chain length and angle of tilt of the component fatty acids present in the TAG

molecules. The short spacings however are independent of the chain length. According to the

hydrocarbon subcell packing the polymorphs are classified into three crystallographic types: α, β’and

β [4, 5, 6].

Literature review

9

The α polymorph is characterized by a hexagonal packing of the chains, there is no angle of tilt and the

chains are far enough apart. In this way the zigzag nature of the chains does not influence packing. In

XRD pattern this polymorph is characterized by one strong short spacing line at 4,15 Å. For the β’

polymorph the chain packing is orthorhombic and perpendicular with an angle of tilt between 50° and

70°, so touching chains are out of step with each other and cannot pack closely. The β’ form can be

seen in the XRD patterns by two short spacing lines at 3,7 Å and at 4,2 Å. The β form has a triclinic

packing in which adjacent chains are parallel to each other and thus pack tightly together. The angle of

tilt is the same as for β’. It is characterized by a strong lattice spacing line near 4,6 Å and a two less

strong lines around 3,6-3,8 Å. This is illustrated in Figure 3 [1, 4].

Figure 3: The sub cell structures of the three most common polymorphs in triacylglycerols [4]

These polymorphs differ in stability, density, melting point and melting enthalpy. The α-form is the

least stable and has the lowest density, melting point and melting enthalpy; whereas the β-form is the

most stable and has the highest density, melting point and melting enthalpy. The β’-form shows

intermediate properties [4, 5]. The layered structure is measured with XRD at long spacings; the lines

that are observed are related to the thickness of the layers formed by side-by-side arrangement. This

layer thickness depends on the length of the molecule, and hence on the number of carbon atoms in the

fatty acids chains, and on the angle of tilt between the chain axis and plane of the methyl end groups

[4, 5].

TAG are aligned from head to tail and show a chair shaped structure wherein the fatty acid on the 2-

position forms the back of the chair. A difference can be made between a 2L or 3L packing depending

on the fact if there are pairs of two or three fatty acid chains. The 3L packing is found when the fatty

acids chains are mixed (large difference in number of carbon atoms of the different chains or saturated

and unsaturated fatty acids are part of the same TAG). This is illustrated in Figure 4 [4, 5].

5.2.4.2 Phase transitions

The relative stability of two polymorphs and the driving force for phase transitions between them at a

constant temperature are determined by their respective Gibbs free energies, with the most stable

polymorph having the lowest Gibbs free energy. The latter is highest for α and the lowest for β and

intermediate for β’. This is a consequence of the higher heats of fusion of polymorphs with a higher

melting temperature. So the stability increases from α to β’ and to β. These three can all directly

crystallize from the melt, when they are heated above their melting point they will return to the liquid

phase. Interpolymorphic transitions , however, are mostly monotropic or irreversible and display first-

order kinetics [1, 4, 5].

A metastable crystal can transform to a more stable one by either the rearrangement of its structural

unities until a complete transformation occurs (solid phase transition) or by melting and

recrystallization ( melt-mediated phase transition). If more than one phase transition from a less stable

to a more stable is possible, the closest more stable phase usually will be formed and not the most

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stable. The rate of these transitions can range from almost instantaneous to very slow for some solid-

state transitions. [1, 4, 5].

Figure 4: Arrangement of triacylglycerols in the crystalline phase: double and triple chair [4]

5.2.4.3 Polymorphic behavior of the studied triacylglycerols

Monoacid TAG such as PPP and SSS

The polymorphic behavior these TAG with an even number of carbon atoms are well represented by

the behavior of SSS and PPP. They show the three basic polymorphic forms, with the exception that

multiple β’-form can occur. The α-form crystallizes upon cooling from the melt, the β’- and β-forms

can be obtained via melt-mediated transformation. The crystal packing of the latter depend on whether

the number of carbons in the chain is even or odd. Three different submodifications of the β’-form

were reported in even carbon number shorter than C16, the third melting closest to the β-form. The

difference in melting points decreases with increasing chain length. The melting point of the α-form

increases monotonically with fatty acid chain length, those of the β’ and β show fluctuations due to the

odd-even chain length effect [1].

Mixed-acid saturated/unsaturated TAG such as POP and SOS, PPO and SSO

TAG with an unsaturated fatty acids at the sn-2 position and saturated fatty acids at the other positions

(Sat-U-Sat) are the main components of many vegetable fats and oils such as palm oil, particularly as

the fatty acid art the sn-2 position is oleic acid. The double bond gives greater steric hindrance, this

leads to the formation of specific structures to be formed to enable the saturated and unsaturated

moieties to be packed in the same lamella leaflet. The polymorphic structures of Sat-O-Sat (O

referring to oleic acid) are similar except for POP, in this way all the others can be represented by

SOS. The polymorphic structures of POP and SOS are given in Figure 5. As can be seen from the

figure an extra intermediate phase γ can occur which has a triple chain length structure, wherein the

oleic acid chains pack in a hexagonal subcell while the saturated chain leaflet shows a parallel

packing. For SOS and so most of the SAT-O-SAT the β’-form also has a triple chain length structure,

here the oleic acid leaflet is again in a disordered hexagonal subcell while the saturated chain leaflets

form an ordered O subcell. In the two β-forms both the saturated and oleic acid leaflets pack in an

ordered manner. Between the two there is a slight difference in the length of the triple chain length

structure and in the melting temperature ( 1,5-2,0°C). The triple chain length structure of the β’- and β-

form is due to the presence of the double bond in oleic acid, in this way the oleic acids are packed

together and separated from the saturated chains. This is not the case for the β’-form of POP because

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the palmitic and oleic acid chains pack to a similar length due to the kink in the oleic acid chain,

resulting in a weaker steric hindrance to the formation of a double chain length structure [1].

Figure 5: A structural model of the polymorphic behavior in Sat-O-Sat TAGs represented by the behavior of POP

and SOS [4]

For mixed saturated/unsaturated triacylglycerols, the β’-form is usually the most stable if the TAG is

asymmetrical (two saturated acid or two unsaturated acids occupy the 1,2- or 2,3-positions), for

example PPO and SSO. This is due to packing requirements for the β’-form are less stringent than for

the β-form. Both PPO and SSO only show two polymorphs being α and β’[4]. The β’-form is usually

the most functional in fat products due to its smaller size and needle shaped morphology. The stability

of this polymorph is influenced by:

Fatty acid chain length and diversity

TAG carbon number and diversity

TAG structure

Presence of a specific TAG

Level of liquid oil present in a fat system

Temperature fluctuation during storage

These factors in combination with the processing history affect the polymorphic behavior of a fat

system [6].

5.1.5 Compound crystals

Compound or mixed crystals contain two or more different molecular species, if these are similar in

size, shape and properties the formation of compound crystals is favored. TAG mixtures easily form

these crystals in the α polymorph since this is the least dense packing mode whit some freedom of

movement and so allows the different molecules to fit into the same lattice. In the β'-polymorph the

compound crystal formation is only possible for similar TAG’s due to the denser crystal lattice, in the

β-polymorph it will only occur for very similar TAG’s in restricted compositional ranges. Most

compound fat crystals are not stable and tend to rearrange themselves into purer crystals often

accompanied by a polymorphic transformation. Extensive compound crystal formation can have

several consequences: the melting range is more narrow; the melting temperature at which most of the

fats melts depends on the crystallization temperature; slow cooling leads to less solid fat compared to

quick cooling to the same final temperature; unstable polymorphs tend to persist much longer [5].

5.1.6 Microstructure of fat crystal networks

The microstructural level of a fat crystal network can be defined as those structures that have a size

between 1 and 140 µm. The lower range of the microstructural level is characterized by crystallites,

while at the upper range clusters of crystals (aggregates) appear. Microstructural development consists

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of the following stages: aggregation, network formation and sintering. These are illustrated in Figure 6

[4, 5].

Figure 6: Various stages in microstructural development: aggregation, network formation [4]

Once the fat crystals obtain a certain minimum size (roughly 0,1µm), they will attract each other by

Van der Waals forces. The only repulsive force is a hard core repulsion, which only operates at very

short distances between the crystals (<0,5µm). The random aggregation of particles that meet each

other via Brownian movement (perikinetic aggregation) and subsequently stick together gives rise to

the formation of fractal aggregates. A specific property of such fractal aggregates is that their structure

is self-similar, implying that they have on average the same structure when observed at different

magnifications. It takes 10 to 100 seconds to form aggregates of a few particles [4, 5].

From the moment the volume fraction of the particles in a fractal aggregate approximates the volume

fraction of primary crystals in the system, the aggregates start to hit each other and so forming a

continuous network. This network is responsible for the elastic properties of fats, whereas merely a

solution of fat crystals or non-touching aggregates will just have a higher viscosity than that the oil.

The formation of such a network or gel takes 2 to 5 minutes, this is at a very low fraction of solid fat

(1%) [4, 5].

When the primary network is formed the major part of the fat still has to crystallize. This additional

crystallization leads to the compaction of the aggregates that comprise the primary network. Whether

the fractal nature of the network still exists at these higher solid:liquid ratios is controversial. Next to

this compaction the further crystallization also leads to sintering. This is the formation of solid bridges

between aggregated crystals and aggregates. Sintering of two crystals will happen if some TAG

molecules are incorporated in the lattices of both crystals. The chance of this occurring is larger at

crystal surfaces that have defects due to lattice mismatches and may be related to the occurrence of

compound crystals. Hence it is more likely that sintering takes places in fat that contain a whole bunch

of different TAG. It has also been shown that polymorphism has an influence on the occurrence of

sintering. The latter only takes place when the outer part of the crystals and the bridging molecules

have the same polymorphic structure [4, 5].

The bonds that construct fat crystal networks can be divided into primary (crystal bridges) and

secondary bonds (van der Waals forces). These primary bonds can be destroyed due to mechanical

treatment, resulting in softening of the fat. The firmness however can subsequently increase again due

to the reorganization of the fat crystals into a network stabilized by weak van der Waals forces and

slow (re-)crystallization can lead to the formation of new primary bonds [5].

The microstructural level has a huge influence on the macroscopic properties of fat products since it is

the closest level of structure to the macroscopic world. The nature of the fat crystal network, this

includes the spatial distribution, the number, size and shape of the constituting microstructural

elements can be seriously modified by changes in the crystallization conditions. The properties of a fat

crystal network, and in this way the macroscopic properties of the final product, are easily changed

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with processing conditions. These processing conditions include the cooling rate, crystallization

temperature, agitation speed and storage time [5].

5.1.7 Mathematical models describing fat crystal networks

The macroscopic characteristics, such as spreadability of many fat containing food products are

depended on the three-dimensional fat crystal network that is formed within the finished product.

Prediction of the properties of fat crystal network is therefore of major importance. To accomplish

this, it is necessary to characterize and define different levels of structure present and to determine

their respective relations to the macroscopic properties. Several mathematical models to describe the

relation between the elastic modulus and the network structure have been developed. Best known are

the linear model and the fractal model [5, 7, 8].

5.2 Fat modification techniques

In our current society the issue of healthy food products has become tremendously important. For fat-

bases products this has translated itself in a demand for product with a reduced amount of saturated

fatty acids and trans fatty acids. A lot of research has been done to achieve this while maintaining the

desired product properties The latter is the real challenge since aroma, texture and mouth sensations

are strongly depended on the fat content of the product. For this purpose fat modification techniques

are researched extensively. The combination of these techniques also leads to a greater variety of hard

stocks with a wide range of physical properties. The four basic fat modifications techniques are

hydrogenation, interesterification, fractionation and blending [9, 10].

5.2.1 Hydrogenation

Hydrogenation of oils and fats has been used from the beginning of the twentieth century to produce

hardstocks of a great number of mainly liquid oils. Hydrogenation involves the addition of hydrogen

atoms across unsaturated double bonds causing saturation of TAG. This saturation leads to the

formation of more rigid structures due to the new molecular configurations. Depending on the natural

occurring starting point of unsaturation of the oil, the degree of hydrogenation will lead to a more

saturated fat with a higher melting point then the starting material. This process however can lead to

the formation of trans fatty acids. The degree of hydrogenation influences the degree of saturation and

so the SFC. However this aspect must be balanced out to account for the trans fatty acid formation [9,

10].

5.2.2 Interesterification

This process is a rearrangement of the distribution of the fatty acids within and between TAG. The

latter can be done chemically or enzymatically and in a random or controlled matter. It results in a

change of the fatty acid distribution, but the actual fatty acid composition remains the same. The net

effect of this rearrangement is the production of oils that require a certain semi-solid-plastic range

having different levels of structure, each influencing the macroscopic properties of the material [9, 10].

5.2.3 Fractionation

Fractionation is the controlled crystallization of a TAG that results in the separation of a solid phase

(stearin) and a liquid phase (olein), these in turn can be further fractionated. So fractionation comprises

of two steps: a crystallization to produce solid crystals in a liquid matrix and the subsequent separation

of this crystals from the liquid matrix. A fat is completely melted and then slowly cooled to below its

melting point. Gentle or no agitation is applied to encourage the formation of large crystals that can be

easily separated. The crystals are formed by the TAG with a higher melting point than the tempering

temperature. The last step is the separation of the liquid and solid fraction by filtration.

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This results in a liquid and a solid fraction with different physical and chemical compositions. Today

the most important oil in terms of fractionation is palm oil because of its unique fat profile that can be

broken down in individual fractions and subfractions thereof [9, 10].

5.2.4 Blending

The blending of different vegetable oil types from different sources is an efficient alternative for

hydrogenated vegetable oils and it can provide the appropriate physic-chemical properties and

nutritional requirements that are demanded. Another benefit of this technique is that no chemical

modification is necessary and so it answers more to the consumer-driven trend of “all things natural”.

The process of blending is one of trial and error and manipulation with the oil blend is made until a

suitable SFC of melting profile is found. The conditions during the blending operation are important.

For blending it is required that the temperature should be high enough to ensure that all components

are liquid, next an effective agitation is also necessary and last a certain time period is required for the

blend to become homogeneous [9].

5.3 Phase behavior The macroscopic and mechanical properties of a fat are determined by its phase behavior. A natural fat

is a mixture of TAG, each having its own polymorphism and melting behavior. These fats cannot be

considered in terms of their component TAG, but only in terms of their phase behavior [4].

A phase is a state of matter that is homogeneous and separated from another phase by a definite

physical boundary. It is defined fully by its composition, temperature and pressure. The latter can be

ignored for most practical purposes in food products. A natural fat always contains at least two phases:

a liquid and a solid. Usually there is only one liquid phase, but several solid phases present at the same

time. In the liquid state the miscibility of the TAG is almost ideal, meaning no heat or volume changes

occur upon mixing and the Hildebrand solubility equation applies. In real fats TAG also mix in the

solid state to form solid solutions or mixed crystals. The latter is an intimate mixture of two or more

components in the solid state such that neither component can be easily distinguished [4].

5.3.1 Phase diagrams

Phase diagrams show the temperature in function of the composition. They are drawn up to obtain

information on the solid phase behavior, especially on the miscibility of the components in the solid

state. Therefore the mixing behavior in the liquid state must be known, it is generally accepted that the

liquid of a TAG mixed system can be treated as a close approximation to an ideal mixture. For these

systems the equilibrium solid phase properties can then be derived from phase diagrams. Most studies

have been performed on binary mixtures. For the latter five types of phase diagrams have been

observed and are shown in Figure 7[4].

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Figure 7: Phase diagrams of binary mixtures of TAG's, (a) monotectic, continious solid solution, (b) eutectic, (c)

montectic, partial solid solution, (d)molecular compound and (e) peritectic [4]

In Figure 7a the TAG A and B have similar properties (melting point, molecular volume and

polymorph), they mix to form a continuous solid solution. For Figure 7b A and B are less similar and

the solubility of one in the other is limited, thus leading to a mixture of solid solutions and a sharp dip

at the interruption of the liquidus line at the eutectic point. These eutectic systems tend to occur when

components differ in molecular volume, shape or polymorph but not much in melting point [1, 4]. In

Figure 7c the eutectic system shifts to a monotectic as the differences in melting point of the TAG

increase. In such cases the solid high melting component dissolves a substantial quantity, mostly 20 to

30% of the low melting component. A and B can combine to form a special mixture called a molecular

compound (M), that behaves like a new, pure TAG with unique properties that differ from its

component TAG. This is shown in Figure 7d. This diagram resembles the eutectic phase diagrams

(Figure 7b) placed side by side. In Figure 7e a peritectic system is shown, this only occurs in mixed

saturated/unsaturated systems where a least one TAG has two unsaturated acids [1, 4].

The above binary phase diagrams can display only the properties of a two component mixture, for real

fat a multicomponent phase diagram with extra axes for each component TAG above two would be

necessary to display its properties. Mixtures of fats differ from mixtures of pure TAG. A real fat

doesn’t have a unique melting point (no temperature at 0 or 100% where liquidus and solidus or

solindex lines meet). Also there is often a eutectic minimum in the liquidus curve but no precise

eutectic point [1, 4].

Normally phase diagrams describe fat systems at equilibrium and represent them in their most stable

polymorphs, but in practice it takes some time to reach equilibrium in the solid state. So an individual

phase diagram should be considered a snap shot at a given time. Last it should be noted that the phase

behavior of two fats may differ for different polymorphs [4].

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5.3.2 Phase behavior of PPP/POP

In Figure 9 the phase diagrams of POP/PPP mixtures for metastable α and most stable β polymorph are

shown. It can be seen that a monetectic phase is formed for α in the whole POP concentration range.

The solubility of POP in the α-form of PPP is around 10%. In the PPP rich region the α forms of POP

and PPP were in the solid state below 20°C, upon heating both transformed to the β-form. However,

when looking at the POP-rich region a different behavior can be seen. POP shows a successive α-β’-β

transformation [2, 11]. The PPP/POP binary system reveals immiscible montectic properties in the

metastable and stable forms. One should indeed aspect that the PPP/POP mixture is monotectic

because the difference in melting point is 30°C and also because of their different chain length

structure (POP β2 has a triple while PPP β has a double).

Figure 8: (a) phase diagram of α forms and (b) phase diagram of POP–PPP mixture. [2]

If the POP ratio is above 50% the polymorphic transition passes from α to β through the intermediate

β’ for POP and PPP while if the POP ratio is lower than 40% an α-β solid state transformation was

observed. The latter is in contrast to the α-β transformation in pure PPP, in which the transformation is

melt-mediated and sometimes happens through β’. Therefore it is assumed that the exothermic α-β

transformation of PPP involving intersolubilized POP may be caused by the lattice instability of α.

The latter enables a conversion to β without melting. Another interesting fact is the difference in the

solubility of POP in PPP between the α- and β-form. In the α-form it is 10% while it is 40% for β. This

could be a consequence of the long incubation that was carried out for β and quenching that was only

applied for α. If α would be incubated the solubility might increase, but this cannot be realized because

rapid transformation from α to more a stable polymorph will occur [2, 11].

5.3.3 Phase diagram of POP/PPO

In Figure 9 the phase diagram of the PPO/POP mixture is shown, in which it can be seen that a

molecular compound is formed. The formation of such a molecular compound is due to specific

interactions. The two TAG display a synergistic compatibility and pack more easily together than on

their own. These compounds consistently form double chain length structures in the metastable and

stable phases, this in contrast with pure TAG that have a triple chain length structure in stable

polymorph. Molecular compounds also crystallize faster than the pure components of the same

polymorph [1, 12].

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Figure 9: Phase diagrams of (a) stable forms and (b) metastable forms of PPO/POP mixtures (C represents molecular

compounds) [1]

In Figure 9a it can be seen for the stable forms that montectic phases are formed between the

compound (βC) and POP (βPOP). βC is only formed at a concentration ratio of 1:1. When the PPO

concentration is above 50% and the temperature is below 31°C (melting point βC) β’PPO and βC are

formed. Above 31°C β’PPO and liquid are present. It is rather curious that the melting point of βC does

not increase without displaying eutectic behavior between the compound and each pure component. In

Figure 9b it can be seen that also metastable forms (αC and β’C) of the molecular compound are

formed. Also here the monotectic nature is obvious, namely αC and β’C are immiscible with the

corresponding forms of POP and PPO [1, 12].

5.3.4 Phase behavior of SOS/SSO

The combination of two TAG that contain one unsaturated fatty acids is less problematic since like

chains can arrange themselves together. Sometimes two TAG display a synergistic compatibility and

pack more easily together then on their own. This is the formation of a molecular compound, this

happens at 50:50 ratio of the two components. The latter is observed for SOS/SSO and POP/PPO (see

section 1.6.4) [1]. From the phase diagram in Figure 10 it can be seen that at low SSO concentration

and a temperature above 39°C the β-form of SOS is present together with a liquid phase. Just as for

POP/PPO monotectic behavior between the molecular compound and SOS can be seen. At high SSO

concentration and at a temperature of 40°C the β’SSO and βc are present. While at a temperature above

40°C β’SSO and a liquid phase can be observed. At the 50:50 ratio the molecular compound is present

in the β-form [13].

Figure 10: The phase diagram of SOS/SSO [13]

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5.4 Storage

During production of semisolid food products fat crystals are formed. These bulk crystallization

processes are sensitive to several production parameters such as temperature, shear forces and

composition of the fat phase. After the production several crystallization processes can occur such as

nucleation of new crystals, crystal growth, Ostwald ripening, polymorphic transition, migration of oil

and migration of small crystals. Ostwald ripening is the process where small crystals dissolve due to

their higher solubility and the dissolved material is then transported to the larger crystals. This gives

rives to larger crystals, which give looser network with loss of consistency as result. Polymorphic

transitions can give rise to a grainy structure in margarine due to formation of β-crystals. Whereas

post-crystallization crystal growth can lead to the formation of solid bridges( sintering) in narrow gaps

of the fat crystal network. The latter is formed due to mutual adhesion of fat crystals in oil. These fat

crystal bonds can be dived in to primary and secondary bonds. The first are the so called solid bridges,

these are very strong and dissociate upon mechanical work. The last are weaker and exist even after

mechanical softening. Johansson and Bergenstahl [14] considered the formation of bridges as an

additional adhesion force in fat dispersions similar to water bridges in air. This adhesion determines

the structures formed and so the texture and consistency of the resulting products. Therefore,

knowledge of adhesion sources provides a method to adapt these parameters [14].

Johansson and Bergenstahl [14] found that during crystallization of TAG components of soybean oil,

palm stearin and palm kernel, the following processes can take place: nucleation of new crystals,

crystal growth, and formation of bridges (sintering). The latter can be either a true solid bridge or a

bridge of small flocculated crystal nuclei. All of these processes can occur simultaneously [14].

For the process of sintering to occur the high melting crystal and the bridge must be of the same

polymorphic form and that it were true solid bridges and no bridges off small flocculating nuclei. In

this way, β’ crystals are sinterted by β’ fat bridges and β crystals by β fat bridges. The latter is favored

by slow cooling whereas for β’ rapid cooling is preferred. However, rapid cooling and large

temperature gradients may limit sintering because they promote formation of the undercooled α form

that cannot bridge β’ or β crystals. Last they also observed that a maximum in sintering ability

occurred for an optimal sintering fat concentration. This is due to competition between bridge

formation and other crystallization processes [14].

Johansson [15] also investigated the influence of storage at elevated temperatures. It was stated that

strength of fat crystal networks in oil increases at elevated temperatures by increased adhesion

between partially melted crystals. The combination of the low oil viscosity and strong adhesion leads

to extensive fat crystal flocculation at elevated temperatures. Likely the crystal flocs sinter and so lead

to an increase in thickness of semisolid fat [15].

In sedimentation experiments an increase in sedimentation volume was seen for all the mixtures, so it

must be related to strong adhesion between fat crystals at elevated temperatures and not to interactions

between oil and crystals. By rheology experiments it was seen that the high network strength of

semisolid fats at elevated temperatures is not only due to strong adhesion but also to slow structural

changes that accompany the adhesion [15].

These temperature-induced changes in fat structure are a consequence of the increasing solubility of

fat crystals in oil with increasing temperature, leading to an increase in adhesion between fat crystals,

increased network strength, flocculation, sintering and crystal growth. The flocculation process that

leads to the increased firmness is slow. In flocculated systems crystals surfaces can come together

leading to molecular contact. The formation of bridges of liquid, semi-solid, solid TAG in the vicinity

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between crystals at elevated temperature contributes to the firmness of the samples. Crystal growth of

occurred during storage of the samples (Ostwald ripening). But this alone is not the reason for the

increase in network strength because networks formed by larger crystals are looser than networks

formed by small crystals. Also polymorphic transition of β’ fat to the β form due to warm storage can

occur, but again this alone cannot explain the increased sample firmness. The overall result was that a

dramatic increase in network strength during warm storage was due to processes as flocculation and

sintering [15].

Laia et al. [16] observed that during storage the hardness of some samples increased; a higher hardness

suggests a more structured, denser crystal network. It was also seen that some samples had a same

SFC value but a different hardness. The strength of the crystal network depends not only on the level

of fat crystals (% SFC), but also on the polymorphic behavior and size of the fat crystals. Two

products that have the same level of solid fat may show different crystal network strengths. It was also

observed that the SFC values showed an increasing trend on storage. This indicates a higher degree of

firmness of the samples, this probably due to the rearrangement of the crystals into a three-

dimensional scaffolding network on storage. In fresh samples however fat probably exists as loose

crystal chains which slowly branch due to weak van der Waals attraction forces on setting. This crystal

network is later reinforced by strong primary bonds, which slowly form between the larger crystals

leading to harder samples [16].

Palm oil is often used in the production of shortenings and margarines .Its crystallization behavior is

complicated and slow, it shows post-hardening problems and it tends to harden slowly with time. All

of these pose problems for industrial application. Zaliha et al. [17] showed that after one day of

storage G’’values of the blends were higher than their G values, indicating that the blends were more

elastic than viscous in character and highly structured. These changes in the internal structure of the

crystals may also occur during storage and thus could lead to negative changes in product quality.

During storage, the G’ of the blends increased with storage time, it continued to increase during the

fourth week of storage, even though the maximum in solid levels was already reached after one week

of storage. This implies that the samples became harder or increased crystal networking after the

crystallization process. They concluded that post- hardening had occurred without transformation of

the polymorphic form from β’ to β. Therefore the post-hardening is more likely due to crystal

networking after crystallization. The trapped oil in between the crystals just after the crystallization

process would be crystallized upon storage, which causes the whole system to become harder or

brittle, also the crystal growth could lead to the formation of solid bridges (sintering) in between the

narrow gaps of the crystal network upon storage [17].

Martini et al. [18] noticed that for shortenings formulated with palm oil, palm kernel oil and vegetable

oil such as sunflower and soy bean oil the peak melting temperature increased with storage time and

that there was a fractionation in the thermal profiles. This suggests that low melting TAG melted when

stored at high temperatures and high melting TAG could have crystallized. However, the fractionation

could also be due to a polymorphic transition to the β- form (higher melting point then β’). Also it was

observed that a difference between the enthalpy values at the beginning and at the end of storage. So

the storage conditions influence the types of TAG that crystallize and the amount of crystallized

material. Next to the thermal behavior also the textural behavior was studied and it was found that

storage at higher temperatures increased penetrometry values and that in many cases the shortenings

were to soft so they could not be measured. Last the SFC of the shortenings was studied, here it was

observed that when samples were stored at higher temperatures that the SFC values decreased and that

the opposite happened when they were stored at lower temperatures. By these experiments it was

concluded that temperature fluctuations during storage significantly affected the quality (evidenced by

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melting behavior, SFC and texture) of the shortenings. However the quality is not sole depended on

temperature fluctuations but is also influenced by chemical composition and the presence of

emulsifiers. Also it was concluded that the storage conditions affected the texture and the SFC of the

shortenings. Further it seemed that samples that were highly supercooled during storage became

harder while samples that were less supercooled during storage became softer. Mostly this was in

accordance with the SFC values. There were however exceptions, this is explained since differences in

hardness are not only due to differences in SFC value but also due to the presence of different

microstructures or network structures generated during the crystallization process [18].

5.5 Thesis topic Most of the studies that have been performed focus on the physical behavior of natural fats and oils

and the application of those in industry or they focus on mixture of pure TAG which is not realistic.

Rarely attention is paid to TAG composition and molecular interactions. In this thesis the focus will be

on TAG composition. Therefore blends with a same amount of saturated fats were prepared, that differ

in the amount of symmetric and asymmetric TAG and that are either palmitic of stearic based. In this

way the influence of symmetry and of chain length can be evaluated. The thesis also pays attention to

storage behavior of these blends, next to the two points mentioned above also the influence of storage

temperature and time will be evaluated in the storage experiments [19].

Materials and methods

21

6. Materials and methods

6.1 Substrate The samples under investigation were blends made by mixing different amount of six pure fats and

High oleic sunflower oil (HOSF) in order to make blends with 40% saturated fatty acids. These fats

were palm stearin (PPP-source), fully hardened shea olein (SSS-source), palm mid fraction (POP-

source), dry fractionated mid fraction of interesterified primary palm stearin (PPO-source), shea

stearin (SOS-source) and dry fractionated mid fraction of interesterified shea stearin (SSO-source). All

of these materials were provided by Loders Croklaan N.V. (Wormeveer, the Netherlands).

6.2 Fatty acid and triacylglycerol composition Fatty acid composition was determined using high resolution GC as described by Vereecken et al.[23]

and measurements were made in triplicate.

The separation of TAG components was performed by Loders Crocklaan (Wormeveer, the

Netherlands) using HPLC-chromatography. Since with the previous method the separation of

symmetric and asymmetric TAG was not possible a second method was used to determine the TAG

composition of the blends. The TAG composition of the blends was thus determined via silver ion

HPLC with the method mentioned in Macher et al. [30]. The determination was performed with a

Shimadzu HPLC system (Tokyo, Japan) in combination with an evaporative light-scattering detector

(Alltech-3300, Alltech Associates Inc., Lokeren, Belgium). The evaporative light-scattering detector

conditions were 1.6 L/min for the gas flow rate, 65°C for nebulizing temperature, and 1 for the

acquisition gain. The mobile phases were heptanes (A) and acetone (B) and the flow rate was 1.0

mL/min. Prior to sample injection the column was reconditioned at 98% A and 2% B this for 12 min.

After injection of the sample the concentration of B was increased to 3% in 5 min and kept there for 5

min. Then it was further increased to 10% in 10 min and kept there for 5 min, before it was finally

increased to 80% over 10 min. For the sample preparation first a stock solution of 4mg/ml was

prepared with heptane, which was then diluted to a concentration of 0,5 mg/ml. The analysis was

performed in duplicate.

6.3 Making of the blends The composition of the blends was based on the fatty acid and TAG composition of the starting

materials. All the starting materials were melted in the oven at 70°C until they were completely liquid.

Then the appropriate masses for the blends were weighed into a glass beaker, the blends were then

mixed using a magnetic stirrer equipped with a heating block to ensure no crystallization takes place

during mixing. Once the blends were completely mixed cups were filled with 15 ml of the blends and

6ml of the blends was transferred into NMR tubes for further analysis [20].

6.4 Methods to study fat crystallization For the study of the isothermal and non-isothermal crystallization behavior several experimental

techniques can be used. In this thesis Differential Scanning Calorimetry (DSC), pulsed Nuclear

Magnetic Resonance (pNMR) and X-ray diffraction analysis (XRD) were applied.

6.4.1 DSC

DSC is a thermo-analytical technique that monitors the change in physical and chemical properties of

materials in function of temperature. Two modes of operation can be used isothermal DSC and stop

and return DSC [5, 20, 21].

Materials and methods

22

6.4.1.1 Isothermal DSC

The isothermal crystallization curves were obtained with the method described by Vereecken et al.

[19] using the sample preparation procedure B as described by Foubert, et al. [24]. The measurements

are performed in triplicate. The following time-temperature program was applied: (1) holding for 10

minutes at 80°C to ensure that the sample is completely liquid; (2) cooling at 25°C/min to the

isothermal crystallization temperature either 15°C or 20°C; (3) holding for 180 min at this temperature

and heating at 20°C/min to 100°C [19].

6.4.1.2 Stop and return DSC

Some samples already crystallize during the cooling period making the integration of the

crystallization enthalpy difficult. This can be overcome by analyzing with the stop and return method

as described by Foubert et al. [22] using crystallization temperatures of 15°C and 20°C. The

measurements are performed in duplicate.

6.4.2 Solid Fat Content (SFC)

The solid fat content is the solid-liquid ratio in a fat ranging from 0% for completely liquid to 100%

for completely solid. It thus represents the mass fraction of solids present at a certain temperature. The

SFC is usually determined via pulsed Nuclear Magnetic Resonance (pNMR). A non-isothermal and

non-tempered SFC curve is determined as described by Conde [20] with each measurement being

performed in triplicate. Additionally a non isothermal and tempered SFC curve was also recorded as

described by IUPAC 2.150 serial tempered method in the region of 0°C-60°C with each measurement

performed in triplicate.

6.4.3 Rheology

Rheology can be defined as the science of flow and deformation and it describes how a material

responds to an applied stress of strain. The rheological properties of materials are often determined via

rotational rheometry. In this thesis small deformation oscillatory experiments were performed on a

stress controlled AR2000(ex) rheometer. (TA Instruments, Brussels, Belgium) as described by De

Graef et al. (2006). The following time-temperature program was applied: (1) conditioning for 10 min

at 70°C, (2) cooling to 15°C or 20°C at 5°C/min, (3) holding isothermally for of two hours.

Measurements were performed in triplicate at a frequency of 1Hz and a fixed strain of 4.5*103 [26].

6.4.4 XRD

X-ray diffraction is used to identify the polymorphism of acylglycerols and can thus be used to

identify the polymorphism of the samples. Bragg’s law is used to convert the incident angle θ [°] and

the distance between the reflecting entities dr[Å]:

in which n’ [-] is the order of diffraction, for this work it is equal to 1. Depending on the detection

angle relative to the incoming ray, there is a differentiation between wide angle X-ray diffraction

(WAXD) and small angle X-ray diffraction (SAXS). They give rise to different spacings. The first is

used to identify the lateral packing of the fat crystals (α, β’ and β) and is associated with the short

spacings, while the second is used to identify the longitudinal packing of the fat crystals (2L or 3L)

and is associated with the long spacings [21].

The measurements were performed at the University of Liège, Gembloux Agro Bio Tech, Unité de

Valorisation des bioressources, Laboratoire de Science des Aliments et Formulation (Passage des

Déportés, 2, B-5030 Gembloux ). The measurements were performed with a D8 Advance (Bruker,

Materials and methods

23

Germany) with a TTK 450 (Anton Paar), V4 slit, Copper tube and equipped with a Vantec detector

(Bruker, Germany). WAXD-data were obtained in the region 1-13° (resolution 0.00796°, 30sec) while

SAXS measurements were done in the range 15-27° (resolution 0.0159°, 30sec). The following

temperature program was applied: heating to 70°C, isotherm for 30 seconds, cooling at 25°C/min to

the crystallization temperatures 15°C and 20°C. During the isothermal period a measurement was

executed each 30 s.

6.4.5 Fractal dimension

Maragoni and coworkers [3, 28] applied the fractal concept and proposed the following model to

describe the elastic modulus G’ [Pa] of lipid systems in function of the volume fraction of solids φ[-]:

with D the fractal dimension of the network [-], γ* [-] the deformation at the elastic limit, A the

Hamaker constant [6 10-21 J], d the particle diameter [m] and H0 the hard core distance between

particles. The first part of the equation is also referred to as ψ [Pa] a constant independent of the

volume fraction, but dependent on the size of the primary particles and on the interaction between

them. The volume fraction is sometimes presented as the SFC. Also it was stated that there exists a

direct relationship between the elastic modulus of a fat and its hardness index as determined by cone

penetrometry measurements (Narine and Maragoni). So possibly a relation between SFC and hardness

exists. Such a relation is already been mentioned, however in the ln-ln presentation. The latter gives a

linear relationship from which a slope and intercept can be calculated [27].

In order to determine the fractal dimension hardness and SFC measurements were performed at five

temperatures: 15°C, 17,5°C, 20°C, 22,5°C and 25°C. The principles of the hardness and SFC

measurements are elaborated in sections 6.5.2 and 6.4.2. For the hardness measurements plastic cups

were filled with 15 ml sample (25 in total). The samples were placed in the oven at 70°C to erase all

crystalline history after which they were crystallized in a water bath at 15°C for two hours.

Subsequently hardness of five samples was measured in the thermostatic cabinet at 15°C. The

temperature of the thermostatic cabinet was then raised to 17,5°C. After one hour at this temperature

the hardness of five samples was measured. This procedure was repeated for temperatures 20°C,

22,5°C and 25°C.

For the SFC measurements NMR tubes were filled with 6 ml sample (three in total) and placed in the

oven at 70°C to erase all crystalline history. Similar to the samples for the hardness measurements the

NMR tubes were crystallized in the water bath at 15°C for wo hours after which SFC is measured at

15°C (three repetitions). The temperature of the water bath was then set at 17,5°C and the samples

were kept at this temperature for one hour and the SFC was again measured ( three repetitions). The

last two steps were repeated for the temperatures 20°C, 22,5°C and 25°C.

6.5. Methods to study the storage stability

6.5.1 Microscopic analysis

Sensorial properties of fat containing products are influenced by the microstructure, so knowledge of

the latter is essential to be able to control and predict the behavior of food materials during processing.

Via imaging techniques more insight in can be gained [5, 20]. For the Polarized light microscopy

(PLM) experiments a Leitz Diaplan microscope (Leitz Diaplan, Leica, Germany) equipped with a

Linkam PE 94 temperature control system (Linkam, Surrey, United kingdom) was used. Samples were

imaged with an Olympus Color View camera (Olympus, Aartselaar, Belgium) equipped with Cell D

Materials and methods

24

software (Olympus, Aartselaar, Belgium). Images were taken with a magnification of 10x10 (objective

10x eyepiece 10x) [21].

The samples were melted in the oven at 70°C, then some drops are transferred to the microscope slide

with a pasteur pipette and a cover slip is placed upon the rest plate. Next the samples were put in the

thermostatic for crystallization at 15°C or 20°C during two hours. Images were then taken by using

polarized light microscopy directly after the crystallization and also after storage of the samples at

three different storage temperatures being 20°C, 25°C and 30°C for different storage times (directly

after crystallization, one day, one week and one month).

6.5.2 Hardness measurements

The macroscopic rheological properties of networks formed by lipids are of major importance in fat

based food products, they are dependent on the mechanical strength of the underlying fat crystal

network. Macrostructural properties of fats and oils can be investigated by large-deformation

measurements or hardness measurements. The probe will penetrate the product a constant speed of

10mm/min for a distance of 10 mm, starting once the force of 0,2 N is reached as mentioned by

Foubert et al [29] To ensure the measurement of the hardness at a specific temperature the texture

analyzer is placed in a temperature controlled cabinet (Lovibond, Dortmund, Germany). [5, 20, 21]

The following procedure was applied: plastic cups already filled with 15 ml of the sample (five

repetitions for each sample) were completely melted in the oven at 70°C, then the samples were put in

the water bath at 15°C or 20°C during two hours. Then for direct measurement, the hardness of

samples were measured by the texture analyzer directly after crystallization. And the hardness was

also measured for all combinations of storage times (one day and one month) and storage temperatures

(20°C, 25°C and 30°C).

6.5.3 SFC measurements

The solid fat content provides information to predict the functionality of fat and fat based products. It

can for instance be used as a guideline to judge whether a certain oil, fat or blend is suitable for a

particular application. The SFC is measured via pNMR, the principles of this technique are already

explained in section 6.4.2. [21].

The following procedure was applied: the NMR tubes were previously filled with 6 ml of sample

(three replications for each sample) and put in the oven at 70°C during one hour to erase the crystal

history. Subsequently they were put in the water bath at 15°C or 20°C during two hours. For the direct

measurement the SFC value of samples were measured by the pNMR directly after crystallization.

SFC was also measured for all combinations of storage times (one day and one month) and storage

temperatures (20°C, 25°C and 30°C).

Results and discussion

25

7. Results and discussion The aim of this thesis was to study the effect of the ratio symmetric to asymmetric TAG on the

crystallization properties and storage stability of fat blends. To this end, eleven blends with varying

ratios of symmetric/asymmetric TAG, but with an equal amount of saturated fatty acids (40%) were

constructed. These blends were crystallized at 15°C and 20°C and stored at 20°C, 25°C and 30°C. The

effect of chain length was also taken into consideration by comparing palmitic based blends to stearic

based blends.

7.1 Composition of the starting oils

The six pure oils and fats that were used to prepare the blends were high oleic sunflower oil (HOSF),

palm stearin (Ps PPP-source), palm mid fraction (PMF= POP-source), interesterified palm stearin

(IPs= PPO-source), fully hardend shea olein (fhSHf= SSS-source), shea stearin (SHs= SOS-source)

and interesterified shea stearin (iISHs= SSO-source). The different sources were blended with the

HOSF to obtain fat blends with a saturated fatty acids content of 40%. These raw materials were

analyzed for fatty acid and TAG content.

The fatty acid composition of the different fats and oils was determined via GC (see section 6.2). The

results for the most abundant fatty acids are summarized in Table 1. The TAG composition was

obtained via HPLC (see section 6.2) and is presented in Table 2. From Table 1 it can be seen that Ps is

high in C16:0 and is used as PPP source. PMF and IPs show a high amount of C16:0 and quite some

C18:1c, thus they are used as POP and PPO source respectively. The fhSHf contains a high amount of

C18:0 and is used as SSS source. SHs and iISHs contain a high amount of C18:0 and quite some

C18:1c, so they are used as SOS-and SSO-source respectively. All of the TAG sources are rather

saturated with the SSS-source being the most saturated. The HOSF on the contrary is highly

unsaturated and contains a high amount of C18:1c and therefore has a high MUFA value. Next to the

obvious one can see from Table 1 that fatty acid composition is quite similar for POP and PPO and

SOS and SSO, this is logical since interesterification does not chance the fatty acid composition but

merely the distribution among the TAG.

In Table 2 it can be observed that Ps and fhSHf contain a high amount of trisaturated TAG. PMF, IPs,

SHs and iISHs contain much less SatSatSat but contain a lot of SatOSat/SatSatO being respectively

POP and PPO or SOS and SSO. For all of the starting oils the amount of SatOO/OsatO and

SatLSat/SatSatL is rather low. SatLSat/SatSatL is highest for IPs while SatOO/OsatO is highest for

iISHs. The HOSF contains a high amount of OOO.

Table 1: fatty acid composition (%) of the starting oils

fatty acid Ps PMF Ips fhSHf SHs iISHs HOSF

C14:0 1,4 0,9 1,4 0,1 0,2 0 0

C15:1 0 0 0 0 2,1 1,9 0

C16:0 82,2 59,9 63,3 6,8 4,2 3,7 4,1

C18:0 4,3 5,3 5,2 90,4 59,2 52,6 3,1

C18:1c 9,7 30,8 25,1 0,8 31,5 38,5 83

C18:2 ω-6 1,7 2,5 3,7 0,2 2,8 3,5 8,3

C18:3 ω-3 0,1 0,2 0,4 1,5 1,4 1,4 0,2

others 0,6 0,4 0,9 0,2 -1,4 -1,6 1,3

SAFA 88,4 66,3 70,6 97,4 64,1 56,3 7,3

MUFA 9,7 30,8 25,3 0,8 33,7 40,5 83,3

PUFA 1,9 2,8 4,1 1,9 4,3 5,0 9,4

Results and discussion

26

C14:0= myristic acid, C16:0= palmitic acid (P), C18:0= stearic acid (S), C18:1= oleic acid (O), C18:2 ω-6=

linoleic acid (L), C18:3 ω-3= α-Linolenic acid, SAFA= saturated fatty acids, MUFA=mono-unsaturated fatty

acids and PUFA= poly-unsaturated fatty acids

Table 2: TAG composition of the starting oils

TAG Ps PMF IPs fhSHf SHs iISHs HOSF

SatSatSat 76,1 4,5 13,9 94,8 2,9 5,4 2,9

SatOSat/SatSatO 13,7 82,8 69,2 2,8 84,0 63,5 2,9

SatLSat/SatSatL 2,7 6,7 10,8 0,6 6,8 4,8 0,3

SatOO/OsatO 4,3 3,7 3,8 0,6 4,6 19,9 19,2

SatOL/SatLO/OSatL 1,6 1,0 1,0 0,2 0,6 2,7 1,4

OOO 0,6 0,3 0,2 0,3 0,4 2,6 65,4

>3 d.b. 0,5 0,2 0,1 0,0 0,1 0,8 7,4

Sat= saturated and is either P(almitic) or S(tearic), O= oleic acid, L= linoleic acid, > 3 d.b.= more than 3 double

bonds

7.2 General scheme of the research The aim of the research was to investigate whether fat blends with a different ratio of symmetric over

asymmetric TAG show differences in crystallization behavior and storage stability. Next to the effect

of symmetric/asymmetric ratio also the influence of chain length on the crystallization behavior and

the storage stability was studied. To study the effect of symmetric/asymmetric TAG ratio blends P1

until P8 were compared for the palmitic based blends with the ratio decreasing from P1 to P8, while

S1 was compared with S2 and S3 for the stearic based blends with the ratio decreasing from S1 to S3.

Blend S1 was compared with blend P1 (highest amount of symmetric TAG’s), blend P5 with blend S2

(medium amount of symmetric TAG’s) and blend P8 with blend S3 (lowest amount of symmetric

TAG’s) to investigate the effect of chain length. This is illustrated in Figure 11.

Figure 11: General scheme of the research

Results and discussion

27

7.3 Fat blending

The composition of the blends in this research were calculated by taking into account the general

scheme of the research, the fatty acid composition and the TAG composition of the starting oils (see

Table 1 and Table 2). Eleven blends were made, the first eight were palmitic based (P1-P8) while the

three others are stearic based (S1-S3). The TAG composition is illustrated in Figure 12.

Figure 12: Composition of the 11 blends in terms of the starting oils

7.4.1 Fatty acid composition of the blends

The fatty acid composition of the blends was determined via Gas chromatography (see section 6.2)

and is summarized in Table 3.

The palmitic based blends ( P1-P8) contain all around 33% palmitic acid (C16:0), while the stearic

based blends S1-S3) contain around 32% stearic acid (C18:0) All the blends contain around 55% of

oleic acid (C18:1c) The saturated fatty acid content ranges from 36,3 to 39,2 %, so there is a small

deviation of the preset 40% SAFA. The amount of mono unsaturated and poly unsaturated fatty acids

is again similar amongst all blends and is respectively around 55% and 6,5%.

Table 3: fatty acid composition (%) of the blends

fatty acid P1 P2 P3 P4 P5 P6 P7 P8 S1 S2 S3

C16:0 33 32,1 33,4 33,4 33,7 33,1 33,3 33 4,4 4,2 4

C18:0 4,3 4,1 4,2 4,2 4,1 4,3 4,2 4,2 31,6 32 32,1

C18:1c 55,2 56,6 54,9 55,1 54,7 54,9 54,3 54,1 56,5 56,5 56,7

C18:2 ω-6 5,6 5,7 5,7 5,7 5,8 6 6 6,1 5,6 5,6 5,5

C18:3 ω-6 0 0 0,3 0,1 0,1 0,1 0,1 0,1 0,9 0,9 0,9

Others 1,9 1,5 1,5 1,5 1,6 1,6 2,1 2,5 1,0 0,8 0,8

SAFA 38,1 36,9 38,4 38,5 38,8 38,4 38,6 39,2 36,5 36,5 36,3

MUFA 55,6 56,9 55,0 55,2 54,8 55,0 54,8 54,2 56,5 56,6 56,8

PUFA 6,3 6,2 6,6 6,2 6,4 6,6 6,6 6,6 7 7 6,9

Results and discussion

28

7.4.2 TAG composition of the blends

Discussing the properties of fat blends two types of analytical parameters can be distinguished: type

one and type two parameters. Type one are additive parameters that obey mass balance equations,

meaning that the property of a blend is the sum of the fractional contribution of each component to the

parameter. Examples are: the iodine value, free fatty acids, fatty acid composition and TAG

composition. Type two on the other hand indicates non additive parameters that don’t obey mass

balance equations. Examples are: the melting point, SFC and most physical properties. So in this way

the TAG composition of the blends was calculated based upon the TAG composition of the starting

oils (Table 2). The TAG composition of the blends is presented Figure 13.

Figure 13: TAG composition(%) of the blends (Sat= saturated and is either P(almitic) or S(tearic), O= oleic acid, L=

linoleic acid, > 3 d.b.= more than 3 double bonds)

From Figure 13 it can be seen that all the blends contain about the same amount of trisaturated TAG

(SatSatSat), this is the same for the triolein content (OOO). The amount of POP and PPO or SOS and

SSO (SatOSat/SatSatO) is quite similar for all the blends. Overall the blends have a similar

composition and differ only in the type TAG and the ratio of SatOSat/SatSatO. Blend P1-P8 contain

mainly POP/PPO while the blends S1-S3 contain mainly SOS/SSO.

The actual amount of POP, PPO, SOS and SSO is determined via HPLC (see section 6.2). The results

of the latter are summarized in Figure 14. There it can be noticed that for the palmitic based blends the

amount of POP gradually decreases as was intended through fat blending. For the stearic based blends

this decrease is larger since there are only three blends. The content of OOO and SSS or PPP is for all

the blends similar. It is also clear that the blends used to determine the effect of chain length in the

analysis’s ( P1 and S1, P5 and S2, P8 and S3) have a comparable TAG composition except for the

fatty acids present in the TAG’s (P or S).

To be able to compare the two methods used for investigating the TAG composition of the blends, the

amount of Sat2O based on the HPLC data is calculated by adding the amount of POP and PPO or SOS

and SOS. The latter is presented in Table 4 together with the amount of SatOSat/SatSatO obtained via

calculation based on the composition of the starting oils. It can be seen that there is some deviation

between the measured SatOSat/SatSatO and the calculated, especially for P1 and S1 that were

designed to contain the most symmetric TAG. For the other blends the variation is smaller. When a

Results and discussion

29

paired T-test is executed , the p-value is 0,861. This is indicates that there is no significant difference

between the two methods.

Figure 14: TAG composition (%) with separation of the positional isomers of the blends

In Table 4 the POP/PPO or SOS/SSO ratio was also calculated based on the HPLC data ( this is done

by simply dividing the amount of POP or SOS by the amount of PPO or SSO). It can be seen that only

for blends P8 and S3 the ratio is smaller than 1 and thus only those contain a higher amount of

PPO/SSO compared to POP/SOS. For the blends P6 and P7 the ratio is around 1, this could be

interesting for the further analysis since at that ratio it was already documented several times that a

molecular compound could be formed [1,12].

Table 4: Calculation of Sat2O, SatO2 and the POP/PPO or SOS/SSO ratio

Blend SatOSat/SatSatO

(HPLC)

SatOSat/SatSatO

(calculated)

Ratio POP/PPO or

SOS/SSO (HPLC)

P1 40,3 36,35 46,1

P2 36,1 36,24 71,3

P3 35,9 36,14 6,5

P4 34,6 36,04 7,8

P5 34,9 35,93 2,3

P6 34,5 35,83 1,4

P7 33,7 35,73 1,2

P8 33,2 35,62 0,6

S1 41,2 36,41 190,2

S2 35,3 35,71 3,0

S3 36,9 35,18 0,4

Results and discussion

30

7.5 Crystallization behavior

The crystallization behavior of a fat has a huge influence on its macroscopic properties. It is therefore

useful and important to get some insight in the crystallization mechanism of the blends. This is done

by using pNMR (solid fat curve) DSC, XRD, fractal dimension determination and rheology.

7.5.1 Solid fat content (SFC) analysis

Via pNMR the solid fat content of the blends was measured to analyze their crystallization and

melting behavior. This was done by the non isothermal tempered and non-tempered method (see

section 6.4.2) The result is a curve that shows the SFC in function of the temperature. When the blends

are not tempered both the stable and the unstable polymorphs are formed, this creates a larger

variability in the results. When the blends are tempered, its temperature is kept at 26°C for forty hours

to allow the formation of the more stable polymorphs. During the tempering step unstable crystals

melt and more stable crystals are formed. So at the end only stable crystals should be present. The

tempering method is mentioned in section 6.4.2. Since there was little difference between the curve

obtained via the non tempered and the tempered method and for the reasons mentioned above only the

Solid fat curve obtained via the tempered method is shown in Figure 15.

Figure 15: SFC as a function of temperature

Figure 15 shows that there is little difference in SFC profile between the different palmitic based

blends indicating that the symmetric/asymmetric TAG ratio has little influence on the SFC profile

When looking at the SFC at 0°C all the blends seem to start at the same SFC value except blend P2

,which starts lower. The curves of P6, P7 and P8 (most asymmetric TAG) shows the highest SFC

values in the region between 10 and 30°C, which is not the case at higher temperatures. From 35°C to

60°C their SFC values are even lower than those of for instance P1. The slight difference between the

blends could be due to small difference in melting points between symmetric and asymmetric TAG’s

(melting point of POP 36,6°C comparing to 34,6°C for PPO) [31]. At higher temperatures other TAG

start to melt for instance PPP.

From Figure 15 it can be noticed that also for the stearic based blends the SFC profile is quite similar.

However the difference in SFC at the start is larger and is highest for S3. Which has the highest SFC

values between 0°C and 10°C , from 10°C ward on S2 shows the highest SFC values until 40°C, after

which all the curves fall coincide. The slight difference between the blends could be due to small

Results and discussion

31

difference in melting points between symmetric and asymmetric TAG (melting point of SOS 43°C

(β2),41°C (β1), comparing to 41°C for SSO (β’) ) [13, 31]. At higher temperatures just as with the

palmitic blends other TAG start to melt for instance SSS. S2 has a SOS/SSO ratio of 3 to 1, since the

molecular compound is formed at the 50/50 concentrations, the ratio of compound/SOS became 1/1 .

The 3/1 ratio is close to the eutectic points of the SOS/compound phase (SSO 35%) for the most stable

forms [13]. But the blend is no pure SOS/SSO mixture so other TAG can also have an influence.

When the SFC profile of the palmitic and stearic based blends are compared (Figure 15), it can be seen

that the SFC is higher starting from 15°C for the stearic based blends. The latter can be explained due

to the higher melting point of stearic based TAG, leading to a lower solubility and thus a higher

SFC[31]. The onset of the SFC curve however is higher for the palmitic based blends. Indicating that

less crystalline material is present for the stearic based blends after tempering. Without tempering the

SFC of the stearic based blends increased first before decreasing, indicating a rather slow

crystallization. It could be that the transformation to more stable crystals (what is indented by

tempering) is also slow and this was not yet completed when the analysis of the SFC began.

7.5.2 DSC analysis

Via DSC analysis the crystallization behavior of the blends can be investigated. This was done with

two methods (isothermal and stop and return) and for two isothermal temperatures: 15°C and 20°C.

7.5.2.1 Isothermal DSC

Via isothermal DSC the crystallization range of the blends can be determined. The crystallization

during cooling was not studied [20]. The used method of analysis is found in section 6.4.1.1. The heat

flow in function of the isothermal time is shown in Figure 16 for all blends.

In Figure 16a it can be seen that for the palmitic based blends the curves are close together, initially

there is small shift at higher isothermal times going from P1 to P8. In addition it can be noticed that

the peak becomes less wide from P1 to P8. The faster crystallization for the blends containing more

symmetric TAG could be explained by the higher melting point of POP 36,6°C comparing to 34,6°C

for PPO. In the blends that contain both POP and PPO the molecular compound POP/PPO could be

formed, which has a melting point of only 31,2°C. The lower the melting point, the lower the driving

force for crystallization (driving force is the difference between melting point and crystallization

temperature) [31]. Blend P6 and P7 show a symmetric/ asymmetric TAG ratio around 1/1 so here a

molecular compound could be formed, since for blend P8 the ratio is 0,6 some molecular compound

could be present. In combination with a excess of asymmetric TAG this could explain the slow

crystallization. The broader peaks for the blends with a high amount of POP could be due to the

sequential crystallization of different polymorphic forms [31]. It should also be mentioned that

interactions between TAG other than POP and PPO could influence the crystallization behavior, PPP

for instance forms mixed crystals with POP and OOO, the latter could be present in the blends under

investigation [31].

Results and discussion

32

Figure 16: Isothermal DSC curve at 15°C (left column) and 20°C (right column) for all blends

(a) (b)

(c) (d)

(e) (f)

Results and discussion

33

For crystallization at 20°C (Figure 16b) the same pattern can be observed. However the peaks are less

wide than for the crystallization temperature of 15°C and the peak maxima are lower. The latter could

be due to the higher crystallization temperature and hence lower driving force and thus less crystals

formation. The reduced width of the peaks is a consequence of less sequential crystallization due to the

lower driving force. The crystallization peaks seem to be positioned a bit earlier than in Figure 16a.

This can be explained due to the fact that a higher crystallization temperature less crystallization has

taken place during the cooling period [20]. For the both temperatures only one peak is observed for all

the blends, this does suspect a one step crystallization process in the isothermal period [31]. But since

the heat flow is no longer zero when the isothermal period starts, crystallization has already taken

place during cooling and the crystallization is thus actually a two step process. A two step

crystallization process can be due to a fractionated crystallization or to polymorphic transition from a

less stable to a more stable polymorph. This can however not be concluded from isothermal DSC-

curves.

In Figure 16c it can be seen that only blend S1 crystallizes within the shown isothermal region, the

crystallization of blend S2 and S3 (intermediate and high amount of SSO) happens much later. Hence

the crystallization is much slower. In Figure 16d the same pattern can be observed, only the

crystallization peak of S1 is broader compared to the one in Figure 16c, its maximum is lower than in

Figure 16c and has shifted to higher isothermal times. The latter can be explained again by less

crystallization during the cooling stage [20]. At a crystallization temperature of 20°C the driving force

is lower for crystallization is lower and so the time required for crystallization to occur is longer and

the crystallization peak becomes broader and less high[31]. Both in Figure 16c and d S1 has only one

crystallization peak so a one step crystallization is assumed in the isothermal, but just as for the

palmitic blends crystallization has taken place during cooling and the crystallization actually takes

places in two steps.

In Figure 16e and f the comparision between palmitic and stearic based blends is made. In Figure 16e

It can be seen that at a crystallization temperature of 15°C the crystallization of S1 happens faster, the

peak is much higher and less broad than the one of blend P1. Due the presence of two extra carbon

atoms in the TAG, the stearic based blends have higher melting points. Hence their driving force for

crystallization is much greater and the crystallization is thus faster and more crystals are formed.

While for a crystallization of 20°C (Figure 16f) the opposite can be observed. At a crystallization

temperature of 20°C the driving force is lower for crystallization is lower consequently the time

required for crystallization to occur is longer and the crystallization peak becomes broader and less

high. The area under the curve is still larger than for P1 so more crystals are formed, as a consequence

of the higher driving force due to two extra carbon atoms.

Overall it can be concluded that the blends that contain more symmetric TAG crystallize faster than

those that contain more asymmetric TAG. Based on the isothermal data alone one step crystallization

is assumed for all the blends, however most likely crystallization has already taken place during

cooling and thus a two step crystallization process can be assumed.

7.5.2.2 stop and return DSC

To gain further insight into the crystallization process, the blends were analyzed by stop and return

DSC (see section 6.4.1.2). The melting enthalpy as a function of the isothermal time is presented in

Figure 17.

In Figure 17a it can be seen that the curves for crystallization at 15°C show the same pattern for all

palmitic based blends: a first increase to a first plateau, followed by a second increase to a second

Results and discussion

34

plateau. For all the blends, the curves start at a melting enthalpy that differs from zero, indicating that

crystallization has already taken place during cooling. A slightly faster increase can be observed for

blend P2, while the other blends have a similar speed (curve coincide during the increase). The blends

show slight differences in final melt enthalpy at the end of the isothermal period is, P1 has the highest

value. These differences can be explained by the difference in melting point of POP and PPO. The

slightly faster crystallization of P2 could be explained by the fact that it contains somewhat less POP

than P1 and the seeding effect of PPP happens slightly faster [23]. For crystallization of the palmitic

based blends at 20°C (Figure 17b) it seems that no crystallization has taken place during cooling as the

melting enthalpy starts from zero in the isothermal period. All blends show a similar speed of

crystallization. The end plateau is again only slightly different and this could again be explained by

difference in melting point. For the other blends the plateau are situated at lower melting enthalpies at

20°C than at 15°C, this is explained by less crystallization due to the lower driving force as a

consequence of the higher crystallization temperature.

In Figure 17c it can be observed that for all three stearic based blends the melting enthalpy differs

from zero at the start of the isothermal period and hence crystallization has taken place during cooling.

Blend S2 and S3 show a similar pattern for crystallization at 15°C while S1 deviates. The behavior of

S1 shows more resemblance with the palmitic based blends as it also shows clearly a two step

crystallization process. This two step crystallization behavior is also observed for S2 and S3.

However, S2 and S3 do not reach the second plateau within the isothermal period. It can thus be

concluded that S1 crystallizes significantly faster than S2 and S3 and has a higher melting enthalpy at

the end of the isothermal period. This high end meting enthalpy could be explained by a polymorphic

transformation to a more stable form (stable crystals have higher melting enthalpies) that has already

been completed for blend S1 and is ongoing for blend S2 and S3. It can also be observed that S2

crystallizes faster than S3 and has already a higher melting enthalpy at the end which could be

explained by the difference in melting point of SOS and SSO. The latter as well as the interaction of

SSS and SOS, where SSS might retard the crystallization of SOS in blends that contain more

asymmetric TAG, could explain the large difference between S1 on the one hand and S2and S3 on the

other hand[20,23].

Crystallization at 20°C (Figure 17d) resulted in similar curves as for crystallization at 15°C (Figure

17c) for the stearic based blends. The onset seems differs again from zero, however the onset value is

lower so less cooling has taken place during cooling as for crystallization at 15°C. Compared to

crystallization at 15°C the second increase of blend S1 occurs later and a lower final melting enthalpy

is reached at the end of the isothermal period. For S2 and S3 the second increase already starts around

25-30 isothermal time when crystallized at 20°C whereas at 15°C this did not occur before 50 min

isothermal time. Furthermore, S2 and S3 reached a higher melting enthalpy at the end of the

isothermal period. All blends show a slower crystallization and for blend S1 and S3 the end plateau is

slightly lower than when crystallization took place at 15°C . The above mentioned observation are due

to the lower driving force as a consequence of the higher crystallization temperature. S2 however has a

higher melt enthalpy than for crystallization at 20°C. Due to the lower driving force (higher

crystallization temperature) less unstable crystals are formed and thus for blend S2 the transformation

to more stable crystals has begun more early now than for crystallization at 20°C while for blend S3 it

happens later. At a crystallization temperature of 15°C more unstable crystals are present and thus the

transformation takes place later than for crystallization at 20°C. For Blend S1 this transformation is

again already finished.

Results and discussion

35

Figure 17: stop and return DSC curves at 15°C (left column) and 20°C (right column) for all blends

Based on Figure 17e and f the effect of chain length can be assessed by comparing P1 with S1, P5

with S2 and P8 with S3. The onset is lower for the palmitic blends compared to their corresponding

stearic based blends, which could indicate that for the stearic based blends more crystallization has

taken place during cooling. Blend S1 crystallizes faster than blend P1and has a higher end value of

melting enthalpy than blend P1. This can be explained by a faster crystallization and more

crystallization or crystallization into a more stable polymorph for blend S1. For blends S2 and S3 the

crystallization is faster but the end melting enthalpy is lower than for their corresponding palmitic

based blends. This is explained by the fact that the crystallization of S2 and S3 is not yet finished, the

transformation to more stable crystals(higher melt enthalpy) is still continuing. The driving force for

crystallization is higher for these blends, more unstable crystals are formed so the transformation starts

Results and discussion

36

later. The difference between S1 and P1 is much smaller than the difference between S2 and P5 and S3

and P8.

In Figure 17f the palmitic based blends show a melting enthalpy around zero at the start of the

isothermal period, where as for the stearic based blends this is not the case. The latter still have

crystallization during cooling despite the lower driving force due to the higher crystallization

temperature. The same as in Figure 17e can be observed, but now both blend S2 and S3 do have a end

melting enthalpy higher than P5 and P8, this is here a mere consequence of the less driving force and

hence less crystallization.

It can thus be concluded that blends that contain more symmetric TAG crystallize faster, both for

crystallization at 15°C and at 20°C particularly for the stearic based blends. For the palmitic based

blends the difference are so small it is difficult to see a clear effect. At 15°C all the blends have a

melting enthalpy at the start that differs from zero, while at 20°C this is only the case for the stearic

based blends. The blends S2 and S3 show an ongoing crystallization (no plateau).

Overall it can be concluded that all the blends show a two step crystallization process (two increases).

Whether the second step is crystallization of low melting fractions or a polymorphic transition is to be

determined by XRD experiments,. However the melting profiles from the stop-and-return experiments

can provide a first indication. For the palmitic based blends P1 (a and b), P5 (c and d) and P8 (e and f)

these are shown in Figure 18. For the stearic based blends S1 (a and b), S2 ( c and d) and S3 (e and f)

these are shown in Figure 19.

From Figure 18 it can be seen that for all palmitic blends one low melting peak is present at the start of

the isothermal period. Upon melting this fraction recrystallizes into a higher melting peak. At longer

crystallization times this initial low melting peak decreased and eventually disappeared while at the

same time a second peak with a higher peak maximum appeared. This shift of the peak maxima to

higher temperatures indicate that a polymorphic transition from an unstable polymorph (α) into a more

stable polymorph has taken place. This higher melting peak continued to grow even after the low

melting peak had disappeared, indicating additional crystallization (fractional crystallization) from the

melt into the more stable polymorph. From these melting profiles it cannot be concluded if this more

stable polymorph is β’ or β. For P1 and P5 a higher melting shoulder can be observed. For P1

crystallized at 20°C this shoulder already appeared when α were still present. At this temperature the

high melting shoulder that can be observed during the whole isothermal period s it probably due to

recrystallization of the α polymorphs into a more stable polymorph as well as to crystallization of a

higher melting fraction. After the α polymorph has disappeared the shoulder shifts to slightly lower

temperatures. While for blend P5 the shoulder is more likely a consequence of crystallization of high

melting TAG since the shoulder gets incorporated into the second peak when lower melting TAG start

to crystallize too. In contrast, blend P8 does not show this high melting shoulder when crystallized at

20°C. This shoulder also indicates that high melting TAG crystallize first and seed the crystallization

of low melting TAG. These high melting TAG undergo polymorphic transition (high melting peak).

Thus for the palmitic blends both fractionated crystallization and polymorphic transition occur [31].

The high melting shoulder decreases from P1 to P5 to P8, thus when the symmetric/asymmetric ratio

decreases, the area of the high melting peak seems to decrease while the area of the second low

melting peak increases. It could thus be concluded that for the blends with a low symmetric/symmetric

TAG ratio much more crystallization into the unstable polymorph occurs due to the presence of more

low melting TAG (PPO lower melting point than POP).

Results and discussion

37

Figure 18: melting profiles at 15°C (left column) and at 20°C (right column) for blend P1, P5 and P8

For the stearic based blends the melting profiles are more complex as can be seen in Figure 19. In

Figure 19a for blend S1 crystallized at 15°C first a low melting peak is observed that undergoes

recrystallization upon melting. As crystallization proceeds this first peak disappears and a second peak

is formed at lower temperatures, indicating fractional crystallization of a high melting (first peak) and

a low melting (second peak) fraction. Also for blend S1 a shoulder is observed, which is here again

probably a consequence of fractional crystallization. Thus for S1 the melting profiles do not provide

information on polymorphic transitions. This will be further elucidated with XRD. Similar

observations can be made for crystallization at 20°C (Figure 19b).

Results and discussion

38

Figure 19: melting profiles at 15°C (left column) and at 20°C (right column) for blend S1, S2 and S3

In Figure 19c it can be seen that at the start blend S2 showed three different melting peaks. Thus is

seems that three fractions crystallize or that a recrystallization takes place upon melting. During the

isothermal only the intermediate of these three peaks continues to grow. Near the end a peak a higher

temperatures appeared and the initial peak at lower temperatures decreased. This could be an

indication of polymorphic transition towards the end of the isothermal period. At a crystallization

temperature of 20°C (Figure 19d) only one peak was initially observed. As crystallization proceeded a

second higher melting peak appeared, which could indicate a polymorphic transition or

recrystallization upon melting. Together with the appearance of the high melting peak the low melting

peak increased significantly due to ongoing crystallization.

Results and discussion

39

Figure 19e shows a rather complicated pattern for blend S3 crystallized at 15°C. At the start three

peaks are present, similar to blend S2. During the isothermal period the first and third peak decrease

while the second peak grows and a fourth peak appears. Thus it could be that a high melting fraction

crystallize first in to an unstable polymorph and promotes the crystallization of low melting fractions

and then later undergoes polymorphic transition. It is also possible that recrystallization takes place

upon melting. The pattern observed for crystallization at 20°C( Figure 19f) is the same as for blend S2

(Figure 19d). The difference is that the high melting peak appears later. This can be explained since

crystallization is slower due to lower driving force (more SSO with lower melting point).

The melting profiles of the stearic blends are much more complicated than those of the palmitic based

blends. For all blends, both palmitic and stearic, a combination of fractionated crystallization and

polymorphic transition is observed. Whether the assumption of polymorphic transition is correct will

be verified via XRD in the next section. As only XRD can provide unambiguous information on which

polymorphs are present.

7.5.3 XRD measurements: short spacings (WAXD)

The XRD experiments were performed in order to gain more insight in the crystallization process. Via

XRD one can see which polymorphs are formed and if polymorphic transitions have occurred. The

latter is most important since with DSC it was determined that all blends show a two step

crystallization process, this could be due to fractal crystallization or due to polymorphic transition.

With WAXD analysis the lateral packing of the fat blends can be determined based on which the

polymorphs can be identified (α, β’ or β). The results are presented as a 3D figure, with the peak

intensity on Z-axis, the isothermal time and d on the Y- and X-axis. The transformation of 2θ to dr [Å]

(=distance between the reflecting entities) is done via the formula in section 2.4.4. The identification

of the different polymorphs is possible since each of the subcell packing’s is characterized by a unique

set of x-ray diffraction (XRD) lines in the wide angle region between 3.5 and 5.5 Å .The hexagonal

subcell packing of the α-polymorph exhibits only one strong diffraction line around 4.15 Å, the

orthorhombic subcell packing of the β'-polymorph is characterized by two strong diffraction lines

around 3.7 and 4.2 Å and the triclinic subcell packing of the β-polymorph gives a whole series of

diffraction lines with one prominent line at 4.6 Å and two other, less intense lines around 3.6 and 3.8

Å [4].

The WAXD figures are presented in Figure 20 for all stearic based blends an one palmitic based blend

(P1), since the palmitic based blends all show the same WAXD pattern. The start and final polymorph

for all blends are summarized in Table 5.

In Figure 20a it can be seen that at 15°C blend S1 first has a peak around 4.15 Å, corresponding to an

α-polymorph, which decreases as time increases. As the α-peak disappears, several diffraction lines

appear: a small bump around 3.7 Å, a sharp around 3.8 Å, several bumps between 4.2 and 4,4 Å and

one at 4.6 Å, indicating the presence of β’- and β-polymorphs. Most likely a polymorphic transition

from α to β’ to β occurs. It is also possible that β and β’ crystals are also formed directly from the melt.

At 20°C (Figure 20b) S1 also crystallizes first into the α-polymorph which is transformed into a more

stable polymorph at later isothermal times. This is evidenced by the appearance of a whole range of

diffraction peaks: several less clear peaks between 3.6 and 3.8 Å, one clear at 4.1-4.2 Å and two close

to each other at 4.3-4.4 Å. The α-peak reduces and shifts to 4.2 Å. The peaks between 3.6 and 3.8 Å

and between 4.3-4.4 Å indicate the presence of another polymorphic form or multiple polymorphic

forms. The α peak does not disappear completely within the isothermal period.

Results and discussion

40

Figure 20: Waxd (d values) at 15°C( left column) and at 20°C (right column) for the stearic based blends and one

palmitic based blend

Results and discussion

41

Thus it seems that either a polymorphic transition to β’ takes place that is not completed yet or a

polymorphic transition in combination with a crystallization directly from the melt happens as

mentioned for crystallization at 15°C.

For blend S2 the crystallization behavior is clearer as can be seen in Figure 20c and d. At 15°C one

peak, located at 4.15 Å and thus corresponding to the α-polymorph, remains throughout almost the

whole isothermal period. At the end of the isothermal period the α-peak shifts slightly to 4.2 Å and a

small peak around 3.7 Å appears, indicating a polymorphic transition to the β’-polymorph. At 20°C, it

is clear that a transition occurs from α to β as evidenced by the appearance of a strong diffraction peak

at 4.6 Å and some less prominent signals around 3.6-3.8 Å It can thus be concluded that for S2 the

polymorphic transition from α to β occurs faster at higher crystallization temperatures. This could be

explained by the fact that at lower crystallization temperature more crystallization (both low melting

and high melting TAG) can take place due to the higher driving force in the unstable form and almost

no transition has yet occurred in the period of the measurements, while at the higher temperatures less

crystallization (only high melting TAG) can take place due to the lower driving force and hence

transformation takes place faster.

For blend S3 a similar crystallization behavior as for blend S2 can be observed. At 15°C (Figure 20e)

there is also the persistent α-peak, but here it changes to one sharp peak at 3.7 Å and a broad peak at

4.2-4.3 Å, which is characteristic for the β’-polymorph. At 20°C (Figure 20f) the same as at 15°C is

observed but an additional small peak at 4.6 Å appears. This could indicate that a later isothermal

times a polymorphic transition from β’ to β takes place. At 15°C this last transition is probably not

visible yet, The same explanation for this delay can be given as for blend S2. Furthermore, direct

crystallization from the melt into the more stable polymorph is also possible.

For the stearic based blends it can be seen that different temperatures and the difference in

symmetric/asymmetric ratio lead to a different crystallization behavior. All the stearic based blends

show a polymorphic transition at both temperatures but the speed at which it occurs and into which

polymorph it transforms is different. Blend S1 was the only blend that showed a clear formation of β

crystals (Figure 20a).For all palmitic based blends a polymorphic transition occurs from α to β’ as is

show in Figure 20g and h for crystallization of blend P1 at 15°C and 20°C. A peak around 4.15 Å (α-

peak) appear that this disappears for all the blends around 20 minutes, followed by the appearance of

one sharp peak at around 3.7 Å and two peaks close to each other at 4.2 Å. This corresponds to the fact

that for palm oil β’ is the most stable polymorph [1].

Table 5: start and final polymorph within the isothermal period of the WAXD analysis for all the blends

Blend Start polymorph

(15°C)

Final polymorph

(15°C)

Start polymorph

(20°C)

Final polymorph

(20°C)

P1 α β’ α β

P2 α β’ α β

P3 α β’ α β

P4 α β’ α β

P5 α β’ α β

P6 α β’ α β

P7 α β’ α β

P8 α β’ α β

S1 α β’ and β α α and β’

S2 α α and β’ α β

S3 α α and β’ α β’ and β

Results and discussion

42

From Table 5 it can be seen that for the palmitic based blends the final polymorph is β’ at both

crystallization temperatures while the stearic based blends show more variation in their crystallization

behavior.

7.5.5 fractal dimension

The fractal dimension of the blends was determined to see whether a relation between the SFC and the

hardness could be found. The hardness of a fat is an important property and it has a large influence on

the perceived texture of food products. The SFC influences greatly the mechanical behavior of fats,

but using it this to predict the hardness has already been shown to be unreliable [27].

For all the blends, both the palmitic as the stearic based blends, a linear relationship was found

between the ln of the hardness and the ln of the SFC. The slope and there from calculated D-value is

presented in. This D-value is calculated with the following formula based on the relationship between

the elastic modulus G’ and the solid volume fraction; the weak link regime with d=3 developed by

Shih et al. [3]:

Table 6: Slope and D-value for all blends

When looking at the fractal dimension (D) in Table 6: Slope and D-value for all blends

, it can be seen that this is for all the blends around 2,5. Within the palmitic based blends blend P2 has

the highest D whereas P5 has the lowest. There is no trend according to the symmetric/asymmetric

TAG ratio. Within the stearic based blends S3 has the highest fractal dimension and S1 the lowest. For

the stearic based blends a trend can be observed, the fractal dimension increases with decreasing

symmetric/ asymmetric TAG ratio. When comparing the palmitic based blends with the stearic based

blends, the stearic based blends have a higher fractal dimension than their corresponding palmitic

based blend except for S1.

Narine and Maragoni [3] mentioned that the fractal dimension increases for systems with increasingly

defined microstructural elements that pack in an increasingly ordered manner. This would indicate that

systems with a sharp nucleation step (nucleation takes place in a narrow temperature range) will have

higher fractal dimensions. This is due to the fact that samples with instantaneous nucleation

characteristics will have nucleation sites that are more ordered due to heat transfer considerations. The

latter is because the heat released from nucleation events needs to dissipate throughout the network

and this is most effective if this an ordered array of sites. The subsequent growth of the network via a

mass- and heat-transfer-limited process will also influence the fractal dimension. If the nucleation sites

serve as templates for the growth of microstructural elements, there seems to be more order, whereas if

the growth of the network is not restricted to the nucleation centers, the structure becomes more

amorphous. The mass- and heat-transfer effects are influenced by the processing conditions, i.e.,

temperature history, of the crystallization procedure, and, therefore, fractal dimension is strongly

influenced by processing conditions. The various fractions within the particular fat crystal network and

the temperatures at which and the size of the temperature range over which they crystallize will

therefore fundamentally affect the fractal dimension [3].

Blend P1 P2 P3 P4 P5 P6 P7 P8 S1 S2 S3

d 2,6 2,64 2,54 2,57 2,23 2,5 2,57 2,51 2,51 2,57 2,67

Results and discussion

43

When applying this to the current data this would mean that within the stearic based blends where the

fractal dimension increases with the decreasing symmetric/asymmetric TAG ratio, the nucleation

range becomes smaller with the decreasing symmetric/ asymmetric TAG ratio. Whereas for the

palmitic based blends the blends with the highest symmetric/asymmetric TAG ratio show the highest

fractal dimension and hence have the narrowest crystallization range. This could be explained by the

fact that their TAG composition is less broad then those of for instance blends P5 and P6 that contain

both POP and PPO while P1 and P2 contain almost no PPO.

7.5.5 Rheology

Oscillatory rheology provides information on how the structure is build up in time [26]. The complex

modulus, which is a measure for the rigidity of the network, in function of the isothermal time is

presented in Figure 21. In this figure only one curve is presented for each blend since there is little

variation between the repetitions, however the measurements were performed in triplicate.

Figure 21: complex modulus in function of the isothermal time for crystallization at (a) 15°C and (b) 20°C

In Figure 21a the palmitic based blends show a low complex modulus at the beginning of the

isothermal period, while it is much higher for the stearic based blends. This indicates that for the

palmitic based blends a limited structure development takes place while this is much more pronounced

for the stearic based blends. This is a consequence of the higher driving force for crystallization for the

stearic based blends due to the extra two carbon atoms. For both the palmitic as the stearic based

blends the complex modulus show a slow increase followed by a steep increase. The length and the

slope of this increase however are different. For the palmitic blends this increases is steeper and longer

in contrast to for the stearic based blends. After this sigmoid increase the complex modulus still

increases slightly due to further strengthening of the network as a consequence of ongoing

crystallization and recrystallization or aggregation [26]. When comparing the three palmitic based

blends it can be observed that the speed of network formation is and the end value of the complex

modulus is largest for blend P8 and smallest for blend P1 respectively most asymmetric and most

symmetric TAG). For the stearic based blends this reversed, they are highest for blend S1 (most

symmetric TAG).

In Figure 21b the complex modulus at the start is lower for all blends, bur remains quite high for the

stearic based blends. This indicates that some network formation still has taken place during cooling

for the stearic based blends. The pattern of the increase in the complex modulus is the same as at a

temperature of 15°C (Figure 21a). When looking only at the palmitic based blends, it can be observed

(a) (b)

Results and discussion

44

that their curves are closer to each other and their speed of network formation shows little difference.

For the stearic based blends nothing except for the lower start complex modulus is changed compared

to what was observed at 15°C (Figure 21a).

In Figure 22 the phase angle in function of the isothermal time is shown. Only one curve is presented

for each blend since there is little variation between the repetitions, however the measurements were

performed in triplicate.

Figure 22: Phase angle delta (°) as a function function of isothermal time for crystallization at (a) 15°C and (b) 20°C

In Figure 22a the palmitic based blends P1 and P5 show at the beginning of the isothermal time a

phase angle around 90° which indicates that the sample is liquid at the start of the isothermal period,

blend P8 has a phase angle of 80° at the start, which indicates that some degree of network formation

has already occurred during cooling. The stearic based blends have a low phase angle at the start,

indicating that already a lot of network formation has occurred during cooling. The latter can be

explained by the higher driving force of crystallization for stearic based blends, just as for the complex

modulus. For all the blends a distinct decrease in the phase angle is followed by an increase. This

increase can be explained by the fact that during the crystallization (decrease in phase angle) a

considerable amount of crystallization heat is released causing a temporary temperature rise since this

heat cannot be removed immediately. Due to this temperature rise locally crystals will start to melt and

so loosening the network structure and thus creating a increase in phase angle [26]. After this the

phase angle keeps on decreasing due to continuing crystallization and aggregation. But just as with the

complex modulus a the decrease is longer and happens faster for the palmitic based blends. The

observed increase is also much larger for the palmitic based blends, this a consequence of the fact that

virtually no crystallization has taken place during cooling and all crystallization happens in the

isothermal period, causing a great release of heat. For the stearic based blends a lot of crystallization

has taken place during cooling and most of the crystallization heat was released there. The latter is a

consequence of the higher driving force and also explains the shorter and later decrease.

When looking only at the palmitic based blends in Figure 22a it can be observed that P8 shows the

fastest decrease followed by P5 and P8. The decrease is lowest for blend P1. When comparing the

different stearic blends S1 shows the fastest decrease, followed by S2 and S3, this is in accordance

with the difference in melting point of SOS and SSO.

(b) (a)

Results and discussion

45

In Figure 22b the palmitic blends start again at 90°, while the stearic blends start around 25°, this is a

bit higher than at 15°C (Figure 22a) due to higher crystallization temperature and hence lower driving

force. The same pattern of decrease in phase angle is seen as at 15°C (Figure 22a) for all the blends.

Also here the decrease is faster and longer and the increase higher for the palmitic based blends.

Furthermore, it can be seen that for the palmitic based blends the curves are closer to each other and

almost no difference in speed or length of the decrease is observed. This is also true for the stearic

based blends. It can be concluded that based on the rheological data all the blends show a two step

network formation and that quite a lot of structure development takes place during cooling for the

stearic based blends due to their higher driving force.

7.6 Storage experiments Storage experiments are executed since the hardness, the SFC and the microstructure (crystal

morphology and crystal growth) are influenced by the storage time, the storage temperature and the

crystallization temperature. After crystallization at both 15°C and 20°C all bends were stored at 20°C,

25°C and 30°C to assess the storage stability. During storage the microstructure (PLM), SFC and

hardness (texture analysis) were monitored for all blends. The SFC and the hardness were measured at

two points in time (one day and one month of storage) while microscopic analysis was performed at

three points in time (one day, one week and one month of storage). The effect of storage temperature,

storage time, symmetric/asymmetric TAG ratio and chain length was assessed.

7.6.1 Effect of crystallization temperature

The microstructural level influences greatly the macroscopic behavior of the fat crystal network. The

macrostructure is investigated by measuring the hardness of the different blends. The SFC is a

frequently used parameter in the food industry, it influences the quality of the food products and can

be used to predict the hardness of fats. The method for the hardness measurements is described in

section 6.5.2, the SFC was measured with the method mentioned in section 6.5.3 and the microscopic

slides were analyzed by PLM as described in section 6.5.1. To be able to assess the effect of the

crystallization temperature on the hardness, the SFC and the microstructure of the fat blends; the

hardness and the SFC were measured and the microstructure was evaluated via PLM directly after

crystallization at 15°C and 20°C.

The results are presented in Table 7 for the hardness and the SFC and in Figure 23 for the

microstructure. Statistical analysis (one way anova and tukey test) was performed on the hardness and

SFC data. Via Tukey it was investigated if there was a difference between the palmitic based blends at

15°C and 20°C and between the stearic based blends at 15°C and 20°C.

In Table 7 it can be observed that for all the palmitic based blends the hardness directly after

crystallization is lower when it has taken place at 20°C. This is logical since at higher crystallization

temperatures the driving force for crystallization is lower. When comparing the blends it can be

observed that at a crystallization temperature of 15°C blend P1 shows the highest hardness and P5 the

lowest. The blends P2, P3, P4 and P6 (same letter b in Table 7) show a similar hardness, this is also

the case for blends P7 and P8. At a crystallization temperature of 20°C less variation is observed

between all the blends. At this crystallization temperature blend the highest hardness is observed for

blend P8 while P2 and P7 show the lowest hardness. Furthermore Table 7 shows that for all stearic

based blends except for blend S2 the hardness is lower when crystallized at 20°C, this is due to the

lower driving force. Here the opposite than with the palmitic based blends can be observed the blends

show less variation in hardness when crystallized at 20°C than at 15°C. At 15°C there is almost no

difference in hardness while at 20°C the hardness decreases from blend S2 to S3 to S1. When

comparing the hardness at both crystallization temperatures it can be observed that at both

Results and discussion

46

crystallization temperatures the stearic based blends have a higher hardness than the palmitic based.

This can be explained by the higher driving force due to the two extra carbon atoms present in their

TAG.

Table 7: Hardness and SFC directly after crystallization at 15°C and 20°C for all blends (different letters indicate

significant differences)

Blend Crystallization temperature

15°C 20°C

Hardness(N) SFC(%) Hardness(N) SFC(%

average stdev Average stdev average Stdev average stdev

P1 21,41

a 1,50 38

f 0,26 3,21

oq 0,40 22,07

u 0,55

P2 11,86

b 0,35 34,03

g 0,45 2,16

pq 0,22 18,43

v 0,12

P3 11,93

b 0,85 35,30

h 0,56 2,72

opq 0,30 18,50

v 0,30

P4 11,75

b 0,87 33,47

gi 0,50 2,73

opq 0,28 17,87

v 0,15

P5 9,63

c 0,51 32,47

ij 0,35 3,37

o 0,34 17,87

v 0,40

P6 12,58

bd 0,54 32,70

ij 0,35 3,31

oq 0,43 17,80

v 0,17

P7 14,35

d 0,67 31,63

jk 0,21 2,46

opq 0,33 18,00

v 0,10

P8 14,52

d 0,68 30,73

k 0,50 5,69

r 0,94 18,23

v 0,25

S1 33,73

e 1,16 40,93

l 0,23 19,40

s 1,27 38,73

w 0,45

S2 33,19

e 0,69 41,87

m 0,12 41,28

t 1,25 39,70

x 0,20

S3 33,05

e 1,10 42,57

n 0,47 30,95

t 2,67 37,30

y 0,10

The palmitic blends show a similar SFC (Table 7), except for P1 that has a higher SFC at both

crystallization temperatures. A somewhat decreasing trend can be observed from blend P1 to P8, and

thus from a high ratio symmetric/asymmetric TAG to a low one. This can be explained by the

difference in melting point of POP and PPO. For the stearic based blends the opposite trend is

observed for crystallization at 15°C as the SFC decreases from S3 to S1. At a crystallization

temperature of 20°C the SFC decreases from S2 to S1 to S3. Blend S2 shows both the highest SFC and

hardness is highest for blend S2, thus it seems that for this blend the most crystallization has taken

place. For all the blends the SFC is lower at 20°C since the driving force for crystallization is lower.

The stearic based blends have a higher SFC than the palmitic based blends, but the difference is much

larger when crystallized at 20°C. The latter can be explained since the stearic based blends have a

higher driving force for crystallization since their TAG contain two extra carbon atoms. The effect is

larger at 20°C since their only high melting TAG can crystallize and the TAG present in stearic based

blends are higher melting than those of the palmitic based blends since they contain two extra carbon

atoms.

When comparing hardness and SFC for the palmitic and stearic blends directly after crystallization it

can be seen that the difference is larger for hardness than for SFC. It can thus be concluded that an

increase in hardness is not only due to more crystalline material but also to the microstructural

arrangement of the crystals

Results and discussion

47

Figure 23: microscopic images directly after crystallization for (a-b) P1, (c-d) P5, (e-f) P8, (g-h) S1, (i-j) S2 and (k-l) S3 (scale bar 200µm)

Results and discussion

48

Fat blends can have different crystal morphology under the same storage condition depending on the

rate of crystallization; which is influenced by the crystallization temperature. A blend that has a fast

crystallization will show a dense crystal network with a high amount of crystals. Also when the

crystallization is fast the crystals will be smaller.

In Figure 23 the microscopic images directly after crystallization are shown for crystallization at 15°C

(upper row) and at 20°C (bottom row). By comparing the images at the two crystallization

temperatures it can be concluded that the crystal network is less dense when the crystallization has

taken place at 20°C, which is due to the lower driving force for crystallization at higher temperatures.

Sometimes the formed crystals at a crystallization temperature of 20°C are smaller than those at a

crystallization temperature of 15°C for the palmitic based blends. At a crystallization temperature of

15°C the palmitic blends show different crystal sizes: blend P4 and P5 have larger crystals while blend

P6, P7 and P8 have smaller crystals. For the stearic based blends no difference can be observed

between the two crystallization temperature since the crystals are small and the network is very dense

for all the blends at both crystallization temperatures. The stearic based blends have a large driving

force is and hence these blends show at both crystallization temperatures a very dense network. Blend

S3 has a denser network than S1 and S2.

7.6.2 Effect of storage conditions

Hardness and SFC were measured after one day and after one month of storage as described in section

6.5.2 and 6.5.3. The microstructure was analyzed after one day, one week and one month of storage as

described in section 6.5.1. For both the hardness and the SFC values statistical analysis was

performed. The results of the latter are shown in Table 9 in the appendix.

7.6.2.1 Evolution of the hardness during storage

The results of the storage experiments are presented in Figure 24.In Figure 24a it can be observed that

for all the blends hardness increased during storage at 20°C following crystallization at both 15°C and

20°C except for blends P6 and P7. It seems that upon storage post-hardening has taken place. After the

production several crystallization processes can occur such as nucleation of new crystals, crystal

growth, Ostwald ripening, polymorphic transitions, migration of oil and migration of small crystals

and sintering which is the formation of solid bridges (sintering) in narrow gaps of the fat crystal

network [14]. All blends show a higher hardness after one day of storage the hardness for

crystallization at 15°C compared to at 20°C; this is explained by lower driving force for

crystallization. However, after one month of storage the inverse is observed except for blends P6 and

P7. Blend P1 shows the highest hardness for all combinations of storage time and crystallization

temperatures. For the combination of one day and a crystallization temperature of 15°C pair ways

resemblance is noticed for P3 and P4 (see Table 9), for the combination of one month and a

crystallization temperature of 20°C resemblance is noticed for P6 and P7 (see Table 9). The latter

pairs have a comparable TAG composition (Figure 14) and therefore the same values would be

expected.

From Figure 24b it is clear that hardness decreased during storage at 20°C for both crystallization

temperatures except for blend S3 for which the hardness seemed to have increased slightly. Apparently

no post-hardening occurred upon storage for the stearic based blends. A possible explanation could be

that either crystal mass was lost due to dissolving crystals or that the network strength has decreased

due to Ostwald ripening. For the stearic blends the hardness is lower when crystallized at 15°C than at

20°C after one day of storage (except blend S3), while after one month of storage the inverse is

observed. Blend S2 show the highest hardness for all combination of storage time and crystallization

temperatures, while S1 shows the lowest. The difference between S1 and S3 is not significant after one

Results and discussion

49

day of storage at crystallization temperature of 15°C (see Table 9), but rather large after one month of

storage at both crystallization temperatures. It can thus be concluded that S2, having a symmetric/

asymmetric ratio of 3, is the hardest and that S1 that contains almost no SSO undergoes a drastic

structure loss while this is not the case for the blends that contains a significant amount SSO.

Furthermore, the stearic based blends, as can be concluded from Figure 24a and b, have a higher

hardness than their corresponding palmitic based blends for all combinations except for blend S1 and

P1 for one month storage at both crystallization temperatures. This is again explained by the fact that

more crystallization has taken place for the stearic based blends due to the higher driving force and

even though their hardness has decreased upon storage their initial hardness was much higher as can

be seen in section 7.6.1.

When the hardness after one day is compared to directly at crystallization it is observed that for both

stearic and palmitic blends the hardness is higher after one day of storage when crystallized at 20°C.

So here it seems that post-hardening occurred due to either, nucleation of new crystals, crystal growth

or polymorphic transition. For the crystallization temperature of 15°C this is not so clear as for some

blends the hardness has increased while for others it has remained constant.

In Figure 24c it can be concluded that the hardness is decreases upon storage at 25°C for blend P1, P2,

P4 and P8 for both crystallization temperatures. For blends P3, P5 and P7 this is only observed only

for crystallization at 15°C. In contrast, blend P6 showed an increase in hardness during storage for

both crystallization temperatures (in Table 9 the difference between the two storage times is indeed

significant). When comparing the different palmitic blends it can be seen that after one day of storage

the hardness is similar for blends P3, P4, P5 and P6 for both crystallization temperatures ( as can be

seen in Table 9). When comparing the hardness after one day of the storage to the hardness directly

after crystallization, it can be seen that the hardness has decreased. It thus seems that upon storage

crystals dissolve in to the liquid phase. Since at 15°C much more (both low and high melting TAG)

crystallization has taken place the hardness decrease greatly when stored at higher temperature.

However, for the crystallization at 20°C less crystallization takes place due to the lower driving force

and only high melting TAG can crystallize, which can withstand the higher storage temperature. This

is why hardness has decreased only slightly after one day of storage at 25°C for a crystallization

temperature of 20°C.

In Figure 24d it can be observed that for all the stearic based blends that storage at 25°C resulted in a

decrease in hardness for both crystallization temperatures. This decrease is small for blend S2 and S3

but large for S1. It seems that for blends that contain almost no SSO the network strength decreases

much more than fore those that do contain SSO. The hardness decreased from blend S2 to S3 to S1,

which was also observed for storage at 20°C. It seems that S2 and S2 which contain quite some SSO

have less loss of network strength since directly after crystallization at 15°C the hardness values were

almost equal for the three blends.

Results and discussion

50

Figure 24: Evolution of the hardness during storage at (a-b) 20°C, (c-d) 25°C and (e-f) 30°C

Figure 24e demonstrates a decrease in hardness upon storage at 30°C which is due to loss of the

network as a consequence of crystals dissolving into the liquid phase due to the high temperature. For

one day of storage for all the blends except P4 the hardness is higher when crystallized at 20°C. After

one month of storage the hardness is highest when crystallized at 15°C than at 20°C for blends P1-P5

and lower for blend P6-P8, but the difference is only small since in Table 9 the difference between the

two crystallization temperatures after one day and one month is not significant except for blend P7

after one day of storage). P8 shows the highest hardness for all storage time- temperature

Results and discussion

51

combinations. However its initial hardness after crystallization was not the highest, so it seems that

blend P8 withstands the high temperature the best. But it should be noted that the values of hardness

are very low and almost no difference is observed anymore between the blends after one day of

crystallization as can be seen in Table 9 only blend P8 for both crystallization temperatures and blend

P7 for a crystallization temperature of 20°C is significantly different from the other palmitic based

blends. Blends P3, P4 and P5 show similar hardness after one day of storage for both crystallization

temperatures which is statistically confirmed can be seen in Table 9. For P3 and P4 this is in

compliance with their similar TAG composition. When comparing the hardness after storage at 30°C

with the hardness directly after crystallization it can be seen that the hardness dropped tremendously,

at these high temperature almost all crystals dissolve again.

As for the palmitic based blends, the stearic based blends also show a decrease in hardness upon

storage at 30°C (Figure 24f) except for S3 crystallized at 15°C but statistics show that the difference

between the two storage times was not significant for S3 (see Table 9). This decrease is due to losing

of the network as a consequence of crystals dissolving into the liquid phase due to the high

temperature. The decrease is rather small (~1N) since most of the crystal are already dissolved again,

since it is so small the exception of S3 can just be due the measurements. Statistics show that the

difference is only significant for blend P7 and P8 at both crystallization temperatures and for blend S2

at 15°C (see Table 9). Blend S2 shows again the highest hardness for all storage time –temperature

combinations except for one month at 15°C, but the difference is less the one and could be again due

to the measurement. When the hardness after storage at 30°C is compared with the hardness directly

after crystallization it can be seen that the hardness dropped tremendous just as for the palmitic based

blends, at these high temperature almost all crystals dissolve again.

When comparing the three storage temperatures it can be noticed that for the palmitic blends the

hardness already became rather low (< 5N) at a storage temperature of 25°C (many of the crystals

have dissolved again). At a storage temperature of 30°C the blends have become very soft, thus almost

no crystalline matter is present anymore. The stearic based blends still show a hardness between 15

and 22 N at 25°C storage, which is approximately the half of what is was immediately after

crystallization. At 30°C the hardness has dropped below 8 N. The stearic based seems to withstand the

high storage temperatures better, it must however be mentioned that their initial hardness was off

course also higher. This better resistance is due to the higher initial hardness and due to the fact that

their TAG are more high melting due to the presence of two extra carbon atoms.

7.6.2.2 Evolution of the SFC during storage

Figure 25 presents the evolution of the SFC during storage. In Figure 25a it can be seen that the SFC is

lower for both storage times when crystallized at 20°C. This was also the case directly after

crystallization and can again be explained simply by the difference in driving force. For blend P1-P4

the SFC has remained constant from one day to one month of storage at 15°C, while for blends P5-P8

it has increased slightly ( as can be seen in Table 9). For both crystallization temperatures the SFC

after one day of storage is highest for blend P1. However it should be mentioned that after one day of

storage crystallized at 15°C the SFC values of all the blends are around 30. At a crystallization

temperature of 20°C the SFC decreases from P1 to P4, then increase to P6 and then decreases again to

P8. P6 and P7 show similar SFC for all storage time-temperature combinations, this is logical since

these blends have similar TAG composition( as can be seen in Table 9). When comparing the SFC

after storage with the SFC directly after crystallization one can see that the SFC decreased upon

storage when the blends were crystallized at 15°C and increased when crystallized at 20°C. When they

were crystallized at 15°C, the driving force for crystallization was larger and hence more low melting

TAG could crystallize, that upon storage dissolve again. While when crystallized at 20°C the driving

Results and discussion

52

force for crystallization is lower and so only more high melting TAG can crystallize and less

crystallization has taken place during the isothermal period. Consequently crystallization continued

during storage.

In Figure 25b it can be seen that for the stearic based blends the SFC is either slightly higher or

slightly lower when crystallized at 15°C compared to 20°C, for S2 and S3 it is almost even( as can be

seen in Table 9). The SFC decreased from one day to one month of storage. Blend S2 and S3 show

almost identical SFC although their TAG composition is different (same letters in Table 9). This

similar behavior was also observed with DSC and rheology experiments. S1 that contains almost no

SSO shows lower SFC when crystallized at 15°C (effect largest when stored for one month), but the

same SFC as S2 and S3 when crystallized at 20°C and stored for one day (as can also be seen in Table

9). This was also seen directly after crystallization at 15°C, while for the crystallization at 20°C it

showed that the S2 had the highest SFC, followed by S1 and S3 although the observed difference were

small but significant (see Table 7). When comparing the SFC directly after crystallization with those

of one day of storage one can see that when crystallized at 15°C the SFC showed a small decrease

while when crystallized at 20°C a slight increase could be observed. The same explanation as for the

palmitic based blends in Figure 25a can be given.

In Figure 25a and b it can be observed that the SFC is higher for the stearic based blends than for the

palmitic based blends for all combination of storage time-temperatures.

In Figure 25c it can be seen that also at a storage temperature of 25°C all the blends show a lower SFC

for both storage times when crystallized at 20°C, but this difference is not always significant as can be

seen in Table 9. This was also the case directly after crystallization and it can again be explained

simply by the difference in driving force. For blend P1-P4 the SFC has decreased from one day to one

month of storage for both crystallization temperatures, while for blends P5-P8 it has increased. After

one day of storage blends P3-P8 show similar values as can be seen in Table 9. So it seems that at

storage temperature of 25°C there is no effect of symmetric/asymmetric TAG ratio except for those

who contain almost no PPO. The difference between P2 and P3-P8 is small while the difference

between P1 and P3-P8 is larger. However P1 and P2 have a similar amount of POP, their PPP content

is different so this difference is probably a consequence of the PPP/POP interaction. When comparing

the SFC directly after crystallization with the SFC after storage one can see that for both

crystallization temperatures the SFC decreased upon storage, the effect is larger when crystallized at

15°C and very small when crystallized at 20°C. The decrease is explained by the fact that the crystals

dissolve again when stored at a higher temperature. The effect is larger for lower crystallization

temperatures since here more low melting TAG crystallize that cannot crystallize at 20°C. These

dissolve easily while the high melting TAG that crystallize at both crystallization temperature can

withstand the high storage temperature.

In Figure 25d it can be seen that for all blends the SFC decreased from one day of storage to one

month of storage, the effect is largest for blend S1. Due to the high storage temperature crystals

dissolve. For blend S1 and S2 the SFC is higher when crystallized at 20°C than at 15°C for both

storage times, however the difference is small. When looking in Table 9 it can be seen that these

difference are not significant except for blend S1 after one day of storage. For blend S3 the SFC is

slightly higher when crystallized at 15°C, but this is almost not visible. Blend S1 and S3 shows similar

SFC values, while S2 show slightly higher SFC values for all storage time-temperature combinations

(as can also be seen in Table 9). When comparing the SFC values directly after crystallization with

those after storage one can see that for both crystallization temperatures the SFC decreased upon

storage. Due to the high storage temperature the crystals dissolve again. For the stearic based blends

Results and discussion

53

there is almost no difference in the decrease for crystallization at 15°C and 20°C, since their TAG are

more high melting due to two extra carbon atoms present.

Figure 25: Evolution of SFC during storage at (a-b) 20°C, (c-d) 25°C and (e-f) 30°C

In Figure 25c and d one can see that the SFC is higher for the stearic based blends than for the palmitic

based blends for all combination of storage time-temperatures. This was also the case directly after

crystallization and is a consequence of the higher driving force. And of the fact that the TAG are more

high melting for the stearic based blends and can withstand the higher storage temperature and thus

their SFC does not decrease so much upon storage.

Results and discussion

54

In Figure 25e it can be observed that there is no longer a trend in SFC when comparing the different

storage times for the different blends, for some blends it increases for other it decreases. It should be

remarked that the differences are small since the scale ranges only from 0 to 12 anymore. It can be

seen in Table 9 that indeed no significant difference between the different storage time are found

except for blend P1 crystallized at 20°C. When looking at the effect of crystallization temperature one

can see that here there is still a trend, the SFC is somewhat higher when crystallized at 20°C than at

15°C for both storage times except for blend P7 and P8 after one month storage. This is strange

considering that all the high melting TAG that crystallize at 20°C also crystallize at 15°C and so the

decrease should be equal. A possibility is that at 20°C more crystals are in a stable form (higher

melting point) due to the higher crystallization temperature and therefore the SFC is higher for

crystallization at this high storage temperature. Blend P3 P4, P5 and P6.show similar SFC values,

while blend P1, P2, P7 and P8 deviate (as can be seen in Table 9). However it must be mentioned that

considering the scale of the figure these difference are small. So here again no clear effect of the

difference in symmetric /asymmetric TAG ratio can be seen. When comparing the SFC directly after

crystallization and after storage one can see that SFC has seriously decreased to rather similar values

for both crystallization temperatures despite the initial difference in SFC for the different

crystallization temperatures. This explained by the fact that at this high temperature also the high

melting TAG crystals dissolve.

In Figure 25f it can be observed that for blend S2 there is almost no difference between the two

storage times and the two crystallization temperatures, as can be seen in Table 9, there are no

significant differences except for blend S1 and S3 between both crystallization temperatures after one

day of storage. For blend S1 the SFC is higher after one day of storage when crystallized at 15°C,

whereas after one month of the storage the opposite is observed. For blend S3 the SFC value is higher

when crystallized at 15°C for both storage times. But for both blends the differences between the two

storage times and the two crystallization temperatures are small. In Table 9 it can be seen that the

differences are only significant for blend S1 and S3 between the two crystallization temperatures after

one day of storage. The SFC Values of blend S2 at all combinations of storage time-temperature are

somewhat higher than those of blend S1 and S3, who are similar (same letters for S1 and S3 and a

different letter for S2 in Table 9). When comparing the SFC directly after crystallization and after

storage the SFC has decreased upon storage. This is due to the fact that crystals dissolve again at high

storage temperatures.

When Figure 25e and f are compared the same can be said as when Figure 25c and d were compared.

When all the storage temperatures are compared one can see that the SFC decreases for both the

palmitic based as for the stearic based blends upon storage and that this decrease increases when the

storage temperature becomes higher. This decrease is smaller for the stearic based blends since they

contain more high melting TAG than the palmitic based blends and can thus withstand higher storage

temperatures.

It can be concluded that a clear effect of storage temperature and chain length was observed whereas

for the storage time there was only a clear trend of decrease upon storage of the blends crystallized at

20°C, for the higher temperatures this was not the case.

As for the effect of symmetric/asymmetric TAG ratio there is no clear trend for the palmitic based

blends, what can be said is that the blends that contain almost no PPO show a behavior that deviates

slightly from the other blends that do contain PPO. For the stearic based blends S2 seems to always

have higher SFC value than blend S1 and S3 that show similar values, however S1 contains virtually

Results and discussion

55

no SSO and S3 contains an excess. Blend S2 that has intermediate amount of SSO (ratio of 3)

apparently has the most stable crystals, this could be a consequence of the presence of compound

crystals [1], [12].

7.6.2.3 Evolution of the microstructure during storage

The Effect of the storage temperature and time on the crystal size of the blends is summarized in Table

8.

Storage at 20°C

From the images after one day of storage it can be observed that the palmitic blends P1and P2; P3 and

P4; P5 and P6 and P7 and P8 show similar images. No differences were observed between the two

crystallization temperatures for all palmitic based blends. Blend P1 and P2 show very small crystals in

a dense network, P3 and P4 show larger crystals and in a network that shows some small gaps. Blend

P5 and P6 show slightly larger crystals than P3 and P4 and their network are the least dense and show

the most and the largest gaps. P7 and P8 show again a denser network and smaller crystals. Blend P1,

P2, P7 and P8 show more spiky crystals while blends P3, P4, P5 and P6 have more round crystals. For

the stearic based blends S2 and S3 show similar behavior after one day of storage, a very dense

network for both crystallization temperatures can be observed with very small crystals. Blend S1

shows next to a crystal network with small crystals and some gaps also several big crystals for a

crystallization temperature of 15°C. At a crystallization temperature of 20°C also a network with small

crystals can be seen together with big crystals, but here the underlying network is somewhat denser.

When the images after one day of storage are compared to the images directly after crystallization the

crystals have become smaller for the palmitic based blends. Possible during storage transformation to

a more stable polymorph has taken place. Directly after crystallization there was a difference between

the two crystallization temperatures, after one day storage at 20°C this is no longer the case. This

catch-up is probably due some extra crystallization has taken place upon storage. For the stearic based

blends no change is seen.

No changes in microstructure were observed except for blend S1. In Figure 26 it can be seen that upon

storage blend S1 develops big crystals imbedded in matrix of small crystals. Upon prolonged storage

more of these crystals appear. They show a spherulitic morphology. Which is typical for the β’-

polymorph. The spherulites are formed due slow secondary crystallization so that one layer is

completed before the next layer is created[1]. WAXD analysis indeed showed the presence of a β’-

polymorph for S1 (Figure 20a and d).

(a) (b) (c) (d)

Figure 26: microscopy for blend S1 crystallized at 15°C and stored at 20°C: (a) directly after crystallization, (b) after

one day of storage, (c) after one week of storage and (d) after one month of storage

Results and discussion

56

Storage at 25°C

Here the different blends show again similar behavior in groups: blend P1 and P2; P3-P5; P6 and P7;

S2 and S3. Blend P1 and P2 show small crystals in a dense network. Blend P3-P5 show larger crystals

in a less dense network with some gaps. Blend P6 and P7 have the same crystal size as P3-P5 but their

network is denser. The behavior of blend P8 is intermediate P3-P5 and P6-P7, but lays closer to that of

P3-P5. S1 shows just as for the storage at 20°C big crystals with a underlying dense network with

small crystals. S2 and S3 show a verse dense network with very small crystals.

When the images after one day of storage of both crystallization temperatures are compared it can be

seen that the networks of the blends are less dense when crystallization has taken place at 20°C. Here

the storage temperature is too high to have a catch up like at a storage temperature of 20°C.

When comparing the images directly after crystallization to one day of storage, it can be seen that for

the palmitic based blends the crystal network has become denser and the crystals have become

smaller, this in contradiction to the hardness and the SFC that both decreased upon storage at 25°C

(since smaller crystals lead to a harder network and more crystals results in a higher SFC). For the

stearic based blends no change is observed since the network was already so dense, only for blend S1

a change occurred. Just as at a storage temperature of 20°C next to small crystals also big crystals

arise. Furthermore, it can be observed that the network becomes a bit denser upon storage for both

crystallization temperatures for the palmitic based blends P3-P8. For blend P1 no change occurred

while for blend P2 the crystal network seems to become less dense. For the stearic based blends no

change has occurred accept for blend S1 the amount of big crystals increases.

Storage at 30°C

After one day of storage at 30°C it can be observed that the different blends show again similar

behavior in groups: blend P1 and P2; P3 and P4; P5- P7. Blend P1 and P2 show small crystals in

network with quite some gaps. Blend P3 and P4 show larger crystals in a network with gaps. Blend P5

-P7 show similar images as P1 and P2, but the blend 7 has a slighter denser network. Blend P8 has

smaller crystals and its network is denser. For the stearic based blends there is almost no difference

between the three blends as they show a very dense network with very small crystals. In addition, the

palmitic based blends showed smaller crystals for crystallization at 20°C compared to 15°C whereas

for the stearic based blends no difference were observed.

Upon prolonged storage nothing changed for the stearic based blends, except for blend S1 big crystals

appear at one week of storage and continue to increase in amount up to one month of storage. For the

palmitic based blends the density of the network appeared to decrease from one day storage to one

week of storage, upon longer storage no further changes were observed. As a consequence of the high

storage temperature crystals dissolved again. Upon one day of storage the network has become less

dense for the palmitic based blends compared to immediately after crystallization for both

crystallization temperatures. As a consequence of the high storage temperature crystals dissolve again.

For the stearic based no difference is observed. However, the network was so dense that differences

were difficult to observe.

Results and discussion

57

Table 8: evolution of crystal size upon storage for all blends

Blend Crystallized at 15°C

Storage temperature

20°C 25°C 30°C

direct 1 day 1 week 1 month 1 day 1 week 1 month 1 day 1 week 1 month

P1 ML VS VS VS VS VS VS ML ML ML

P2 ML VS VS VS VS VS VS ML ML ML

P3 ML S S S ML ML ML ML ML ML

P4 L S S S ML ML ML L L L

P5 L ML ML ML ML ML ML L L L

P6 S ML ML ML S S S S S S

P7 S S S S S S S S S S

P8 S S S S ML ML ML S S S

S1 VS VS+B VS+B VS+B VS+B VS+B VS+B VS VS+B VS+B

S2 VS VS VS VS VS VS VS VS VS VS

S3 VS VS VS VS VS VS VS VS VS VS

Crystallized at 20°C

Storage temperature

20°C 25°C 30°C

P1 S VS VS VS VS VS VS S S S

P2 S VS VS VS VS VS VS S S S

P3 S S S S ML ML ML S S S

P4 S S S S ML ML ML S S S

P5 S ML ML ML ML ML ML S S S

P6 S ML ML ML S S S S S S

P7 S S S S S S S S S S

P8 S S S S ML ML ML S S S

S1 VS VS+B VS+B VS+B VS+B VS+B VS+B VS VS+B VS+B

S2 VS VS VS VS VS VS VS VS VS VS

S3 VS VS VS VS VS VS VS VS VS VS

VS= very small, S=small, ML= medium large, L= large, VL=very large and B=big

Results and discussion

58

Conclusion

The microscopy data showed little difference for the stearic based blends since their network is so

dense. For the palmitic based blends differences can be seen. But it should emphasized that

comparison of data obtained with different methods should be done with care since the sample size,

the sensitivity of the technique, etc is different. Furthermore microscopy only allows the visualization

of a limited region of the sample. More importantly crystallization on a microscopic slides is confined

to two dimensions whereas for the hardness and SFC measurements the crystals can develop in three

dimensional space. This could be the explanation why the microscopic data do not always corroborate

with the SFC and hardness . This especially true for the storage at 25°C. For the storage at 30°C the

images show a decrease in density as was also observed for SFC and hardness.

Conclusion

59

8. Conclusion The aim of this thesis is to study the effect of the ratio symmetric to asymmetric TAG on the

crystallization properties and storage stability of fat blends. To this end, eleven blends with varying

ratios of symmetric/asymmetric TAG, but with an equal amount of saturated fatty acids (40%) are

constructed. Eight of these blends were palmitic based and made by mixing different fractions of

palm oil, the other three were stearic based and made by mixing different fractions of shea butter,

symmetric/asymmetric TAG ratio decreased from blend P1 to P8 and from S1 to S3 These blends

were crystallized at 15°C and 20°C and stored at 20°C, 25°C and 30°C. The effect of chain length

is also taken into consideration by comparing palmitic based blends to stearic based blends. so that.

The research consisted of three parts: part one focused on the composition of starting oils and

blends, in part two the crystallization behavior of the blends was investigated while the storage

stability was studied in part three.

In the first part of the research it was confirmed that the symmetric/ asymmetric TAG ratio was

different for the blends and decreased from blend P1 to P8and from S1 to S3.It also confirmed that

the assumption to compare P1 with S1, P5 with S2 and P8 with S3 is was correct, only P2 could

have been chosen too since its ratio was even higher as that one of P1.

In the second part of the research the SFC curves show little difference between palmitic and

stearic blends. The explanation for due to the difference in melting points of POP/PPO or SOS:SSO

seems to apply only at higher temperatures. The solid fat curves of the stearic based blends were

shifted to higher SFC values and higher temperatures higher due to their higher melting points.

The DSC data showed that at a crystallization temperature of 15°C the blends with higher

symmetric/asymmetric TAG ratio crystallized faster for both palmitic and stearic based blends. At

a crystallization temperature crystallization during cooling happened for all blends, while at a

crystallization temperature of 20°C this only the case for the stearic based blends. Blend S2 and S3

had not reached a plateau during the isothermal period, so their crystallization was not yet finished.

The DSC data showed a two step crystallization process for all blends, the presence of a

recrystallization peak for all suggests that it was caused by a polymorphic transition.

The XRD data showed a α to β’ polymorphic transition for all palmitic based blends at both

crystallization temperatures and so supports the assumption made from the DSC analysis. For the

stearic based blends it showed a transition to β’ or β.

The determination of the fractal dimension gave an impression of the order in the crystal network

and the broadness of crystallization range, the higher the fractal dimension the more narrow the

crystallization range and the more ordered the packing in the crystal network is. For the stearic

based blends the order decreased and the broadness of the crystallization range increased from

blend S1 to S3. For the palmitic blend a trend was not really observed only that the fractal

dimension and thus the crystal range was smallest for blends P1 and P2, this is logical since these

contain almost no PPO and thus have a simpler TAG composition.

The rheological data showed that the difference within the palmitic and the stearic based blends

became smaller when crystallization took place at 20°C. For both the phase angle as the complex

modulus the decrease was longer and happened faster for the palmitic based blends. This was a

consequence of the large amount of network formation that had happened during cooling for the

stearic based blends. For the stearic based blends the speed of network formation decreased from

Conclusion

60

S1 to S3 ( with decreasing symmetric/asymmetric TAG ratio). For the palmitic based the effect of

symmetric/asymmetric TAG ratio was not clear.

In the third part the hardness directly after crystallization was lower for all blends when crystallized

at 20°C due to lower driving force. No clear effect of the symmetric/asymmetric TAG ratio was

observed. For the stearic based blends S2 showed the highest hardness for all storage experiments.

The hardness was also higher directly after crystallization and for all storage experiments for the

stearic compared to palmitic based blends. At a storage temperature of 20°C the palmitic based

blends showed post-hardening. At a storage temperature of 30°C the hardness upon storage

decreased for all blends as a consequence of dissolving crystals, this decrease is smaller for the

stearic based blends since their crystals are higher melting and can withstand the high temperature

better.

The SFC analysis does show a decreasing trend of SFC from P1 to P8 en from S3 to S1 directly

after crystallization, the difference between the stearic and the palmitic based in SFC is smaller

than that in hardness, this is because a faster crystallization leads to smaller crystals (higher

hardness). Blends crystallized at 20°C showed ongoing crystallization when stored at 20°C. For the

storage temperature of 25°C and 30°C the SFC was lower after one day of storage than directly

after crystallization as a consequence of crystals dissolving again. The decrease was smaller when

the blends were crystallized at 20°C due to the fact that there only the more high melting TAG can

crystallize. For all storage experiments the SFC values of the stearic blends were higher due to their

higher driving force for crystallization and their better resistance to higher storage temperatures. No

clear effect of symmetric/ asymmetric TAG ratio was observed. For the stearic based blends S2

always showed the highest SFC so a symmetric/asymmetric TAG ratio of 3 leads to the most stable

crystals instead of either an excess of symmetric or asymmetric TAG (respectively blend S1 and

S3).

The analysis of the microstructure showed that directly after crystallization the crystal network was

less dense when crystallization had taken place at 20°C for the palmitic based blends for the stearic

based blends no influence of crystallization was observed. At a storage temperature of 20°C no

difference is observed between the different storage times and for the palmitic based blends the

crystals have become smaller compared to directly after crystallization. The difference that was

present directly after crystallization between the two storage temperatures has disappeared, so it

seems that some catch-up crystallization has taken place. At a storage temperature of 25°C the

difference present between the two crystallization temperatures directly after crystallization

remained. For the palmitic based blend the crystal network became denser and continues to become

denser upon storage compared to directly after crystallization for the palmitic based blends, this is

contrast to the decreasing SFC and hardness. At a storage temperature of 30°C the crystal network

has become less dense compared to directly after crystallization for the palmitic based blends due

to the fact that crystals dissolve again at these high storage temperatures. An odd phenomenon was

observed for blend S1 upon storage at all storage temperatures big crystals formed within the dense

network with small crystals. In all storage experiments the different palmitic based blends showed

differences in size and density but no clear effect of symmetric/asymmetric TAG ratio. Smaller

crystals and a denser network were observed for the stearic based blends directly after

crystallization for both temperatures and in all storage experiments due to the higher driving force

for crystallization. For the stearic based blends the effect of symmetric/asymmetric TAG ratio,

storage time, storage temperature and crystallization temperature was difficult to examine since

their crystal network was so dense due to the driving force for crystallization.

Further research

61

9. Further research Since the storage stability experiments showed an increase in hardness when the crystals were

stored for one day at 20°C, it would be good to investigate what caused this post-hardening. If it is

caused by a polymorphic transition this could be checked by XRD-analysis.

Another lead for further research is the development of large crystals for only blend S1 upon

storage. Maybe a microscopic analysis with a smaller time frame could provide some answers to

when these crystals start appearing.

Next to these small extra experiments that could be performed with the same samples some

research with other blends could also be interesting. Based on the differences that were observed in

all experiments between blend S1 and S2 and S3 it could be interesting to perform the same

experiments on some more blends with a symmetric/ asymmetric TAG ratio situated between that

of S1 and S2 just as for the palmitic based blends.

As was mentioned in the literature review the demand for more healthy fats is becoming more and

more important. Thus it has become a challenge to make products that have less saturated and less

trans fats but still taste, smell,… the same. The TAG composition has an influence on the final

properties and thus by changing the TAG composition the final properties can be changed. This

research showed that a different symmetric/asymmetric TAG ratio led to different behavior. So by

optimizing this ratio the macroscopic properties needed for the production of confectionary

products could be obtained without adding more saturated fatty acids. Next to the

symmetric/asymmetric TAG ratio it could be interesting to see how the saturated/ unsaturated TAG

ratio influences the behavior of fats.

References

62

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Appendices

65

11. Appendices In column 2, 3, 5 and 6 the blends are given the same letter when the difference between them was

not significant, while in column 4, 7, 8 and 9 a * is placed when the difference was significant.

Table 9: significant differences during storage for hardness and SFC

Blend Between

blends at

one day

of storage

Crystalliz

ed at

15°C

Between

blends at

one day of

storage

Crystallize

d at 20°C

Between the

different

crystallization

temperatures

for the blends

after one day

of storage

Between

blends

at one

month

of

storage

crystalli

zed at

15°C

Between

blends

at one

month of

storage

crystallize

d at 20°C

Between the

different

crystallization

temperatures

for the blends

after one

month of

storage

Between

different

storage

times for

crystallizati

on at 15°C

Between

different

storage

times for

crystallizatio

n at 20°C

Hardness: storage temperature of 30°C

P1 a a acd ab

P2 a a b a

P3 a a abc a

P4 a a acd ab

P5 a a abc ab

P6 a a abcd abc

P7 a b * abcd abc * *

P8 b b acd c * *

S1 c c e d

S2 d d f e *

S3 cd c f d *

Hardness: storage temperature of 25°C

P1 ab a * a a *

P2 ab b * b b *

P3 a b a c *

P4 a b a b *

P5 a b c acd * *

P6 a b d e * * *

P7 c b d acd

P8 d a c b * *

S1 e c * e e * *

S2 f d * f f

S3 g e g g *

Appendices

66

Blend Between

blends at

one day

of storage

Crystalliz

ed at

15°C

Between

blends at

one day of

storage

Crystallize

d at 20°C

Between the

different

crystallization

temperatures

for the blends

after one day

of storage

Between

blends

at one

month of

storage

crystalliz

ed at

15°C

Between

blends

at one

month of

storage

crystallize

d at 20°C

Between the

different

crystallization

temperatures

for the blends

after one

month of

storage

Between

different

storage

times for

crystallizati

on at 15°C

Between

different

storage

times for

crystallizatio

n at 20°C

Hardness: storage temperature of 20°C

P1 a a * a a * *

P2 b be * b b * *

P3 cd c * b b * * *

P4 cd d * b c *

P5 b d * b c * *

P6 b be * c d * *

P7 bd be * ac d * *

P8 bcd e * c c * *

S1 e f * d e * *

S2 f g e f * *

S3 ef h e f * *

SFC: storage temperature of 30°C

P1 ac ab abd abd *

P2 b abc ab ab

P3 ac abc cd cd

P4 ac a acd cd

P5 ac ab acd cd

P6 c a cd cd

P7 ab abc acd acd

P8 b bc abd abd

S1 d d * e e

S2 e e f f

S3 d d * e e

Appendices

67

Blend Between

blends at

one day

of storage

crystallize

d at 15°C

Between

blends at

one day of

storage

crystallize

d at 20°C

Between the

different

crystallization

temperatures

for the blends

after one day

of storage

Between

blends

at one

month of

storage

crystalliz

ed at

15°C

Between

blends

at one

month of

storage

crystallize

d at 20°C

Between the

different

crystallization

temperatures

for the blends

after one

month of

storage

Between

different

storage

times for

crystallizati

on at 15°C

Between

different

storage

times for

crystallizatio

n at 20°C

SFC: storage temperature of 25°C

P1 a a acd ac * *

P2 bc b bc bc *

P3 bc b abc abc *

P4 bc b ade ac

P5 bc b * def a

P6 bc b * ef a *

P7 bc b * adef a

P8 b b adef ac

S1 d c * g d * *

S2 e d h e *

S3 d c i d * *

SFC: storage temperature of 20°C

P1 af a a a

P2 bde bef bd b

P3 cdefg ce * bcd c *

P4 bcdeg d * bcd bc *

P5 bcdefg bce * a ac * *

P6 acdefg bf * a a * * *

P7 cdefg bef * a ac * *

P8 bcdefg ce * acd a *

S1 h g * e d * *

S2 i g f e

S3 i g f f * *