Production of Cocoa Butter Equivalent through Enzymatic

Faculteit Bio-ingenieurswetenschappen

Academiejaar 2011 – 2012

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis

Lynn Naessens

Promotor: Prof. dr. ir. Koen Dewettinck Tutor: Sheida Kadivar

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

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis I

The author, promotor and tutor give permission to use this thesis for consultation and to copy parts

for personal use. Any other use falls under the copyright laws: the source must be correctly specified

when results of this thesis are used.

De auteur, promotor en tutor geven toelating om deze thesis te gebruiken voor consultatie en om

bepaalde delen te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder het auteursrecht:

de bron moet uitdrukkelijk en correct vermeld worden als resultaten uit deze thesis worden gebruikt.

Ghent, June 2012.

The promotor The tutor

The author

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis II

Woord vooraf Deze masterproef vormt de afsluiter van 5 jaar studeren aan de faculteit bio-

ingenieurswetenschappen te Gent. De afgelopen 5 jaar waren zeker niet de makkelijkste, maar met

de steun van veel vrienden en familie ben ik ze toch heelhuids doorgekomen. Daarom wil ik zeker

een moment nemen om al deze mensen te bedanken om er voor mij te zijn, altijd te willen luisteren

en mijn gedachten af te leiden van schoolwerk wanneer ik het nodig had.

Vooraleerst wil ik een aantal mensen bedanken voor de hulp en steun tijdens de realisatie van mijn

masterproef. Mijn promotor, Prof dr. ir. Koen Dewettinck, wil ik bedanken om me de kans te geven

deze thesis in de vakgroep FTE te realiseren. Sheida, bedankt voor alle hulp en uitleg bij elk nieuw

experiment. Bedankt dat je elke keer heel snel en met veel geduld mijn vragen beantwoordde en om

zoveel tijd te spenderen in het nalezen van elk deel in deze masterproef. Alle mensen van de

vakgroep FTE wil ik bedanken om altijd klaar te staan met een woordje uitleg bij de vragen die ik had,

de hulp bij het verwerken van gegevens en waar ik bepaalde zaken kon vinden. Ook mijn vele

thesiscollega’s wil ik bedanken voor de vele praatjes tussen de experimenten in.

Ook gaat een woord van dank uit naar het bedrijf Oleon te Antwerpen. Vooral Marjan verdient de

vermelding in deze masterproef voor de tijd die ze vrijmaakte om mij het SPD proces heel geduldig

en vriendelijk uit te leggen.

De jaren op ‘het boerekot’ werden zeker aangenaam gemaakt door de vele nieuwe en ongelooflijk

leuke mensen die ik hier heb leren kennen. De toffe sfeer die we gedurende de afgelopen jaren

gecreëerd hebben, zal mij altijd bijblijven.

Mijn vrienden uit Aalter, ik wil jullie bedanken om mijn gedachten te verzetten elke keer we samen

kwamen om bij te praten of iets leuks te ondernemen.

Ook mijn ouders wil ik bedanken voor de steun die ze mij geven in alles wat ik doe en om mij de kans

te geven alles te doen wat ik maar wil.

Last but not least, mijn vriend Roberto; tijdens het realiseren van de masterproef heb je mij altijd

gesteund en geholpen waar je kon. Nu wordt het tijd dat ik wat meer aandacht aan jou besteed.

Gent, juni 2012

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis III

Table of Contents Introduction ............................................................................................................................................ 1

Literature ................................................................................................................................................ 2

1. Modification of fats and oils ............................................................................................................ 2

1.1 Fractionation ........................................................................................................................... 3

1.2 Hydrogenation ......................................................................................................................... 4

1.3 Interesterification .................................................................................................................... 4

1.4 Blending ................................................................................................................................... 4

2. Cocoa butter .................................................................................................................................... 4

2.1 Chemical properties ................................................................................................................ 5

2.2 Physical properties .................................................................................................................. 5

3. Cocoa butter alternatives ................................................................................................................ 6

3.1 Cocoa butter equivalents ........................................................................................................ 8

3.1.1 Legislation ........................................................................................................................ 8

3.1.2 Sources ............................................................................................................................ 9

3.1.3 Production ..................................................................................................................... 11

4. CBE production .............................................................................................................................. 14

5. Product purification ....................................................................................................................... 16

6. Optimization of the reaction ......................................................................................................... 18

Materials and methods ........................................................................................................................ 19

1. Substrates and enzyme ................................................................................................................. 19

2. Methods ........................................................................................................................................ 19

2.1 Quality of the starting oil ....................................................................................................... 19

2.1.1 Peroxide value (PV) ....................................................................................................... 19

2.1.2 p-anisidine value (p-AV) ................................................................................................ 19

2.1.3 Totox value .................................................................................................................... 19

2.1.4 Acid value and FFA ........................................................................................................ 19

2.2 Chemical composition of the starting oil .............................................................................. 19

2.2.1 Fatty acid profile ............................................................................................................ 19

2.2.2 Triacylglycerol profile .................................................................................................... 20

2.3 The enzymatic acidolysis ....................................................................................................... 21

2.4 Response surface methodology ............................................................................................ 21

2.5 Short path distillation ............................................................................................................ 22

2.6 Fractionation ......................................................................................................................... 23

2.7 Pulsed nuclear magnetic resonance (pNMR) ........................................................................ 24

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis IV

2.7.1 Non-isothermal method (non-tempered) ..................................................................... 24

2.7.2 Non-isothermal method (tempered)............................................................................. 24

2.7.3 Isothermal method ........................................................................................................ 24

2.8 Differential scanning calorimetry (DSC) ................................................................................ 24

2.8.1 Non-isothermal method ................................................................................................ 25

2.8.2 Isothermal method ........................................................................................................ 25

2.9 Polarized light microscopy..................................................................................................... 25

2.10 Statistical analysis .................................................................................................................. 25

Results and discussion .......................................................................................................................... 26

1. Introduction ................................................................................................................................... 26

2. Characterization of HOSO.............................................................................................................. 27

2.1 Chemical characterization ..................................................................................................... 27

2.2 Physical properties of HOSO.................................................................................................. 29

2.2.1 Non-isothermal crystallization and melting behavior as measured by DSC ................. 29

2.2.2 Solid fat content as measured by pNMR ....................................................................... 29

3. Enzymatic acidolysis ...................................................................................................................... 30

3.1 Optimization of the reaction conditions ............................................................................... 30

3.1.1 Reaction time ................................................................................................................ 31

3.1.2 Reaction temperature ................................................................................................... 32

3.1.3 Water content ............................................................................................................... 33

3.1.4 Enzyme load .................................................................................................................. 34

3.1.5 Substrate ratio ............................................................................................................... 35

3.1.6 Improving the yield of the reaction ............................................................................... 36

3.2 Optimization of the reaction parameters by RSM ................................................................ 40

3.2.1 Experimental design ...................................................................................................... 40

3.2.2 Model fitting .................................................................................................................. 41

3.2.3 Main effects and interactions between parameters ..................................................... 43

3.2.4 Optimization .................................................................................................................. 44

3.2.5 Model verification ......................................................................................................... 45

4. Product purification ....................................................................................................................... 46

4.1 SPD ......................................................................................................................................... 46

4.2 Fractionation ......................................................................................................................... 46

4.3 Physical characterization ....................................................................................................... 49


4.3.2 SFC ................................................................................................................................. 52

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis V

5. Chemical composition CB/ CBE mixtures ...................................................................................... 54

5.1 FA profile of CB and CBE ........................................................................................................ 54

5.2 TAG composition ................................................................................................................... 54

6. Physical characterization ............................................................................................................... 57

6.1 Non-isothermal crystallization and melting behavior ........................................................... 58


6.1.2 Solid fat content as measured by pNMR ....................................................................... 59

6.1.3 Isothermal diagram ....................................................................................................... 62

6.2 Isothermal crystallization ...................................................................................................... 63

6.2.1 Isothermal crystallization as measured by DSC ............................................................. 63

6.2.2 Isothermal crystallization as measured by pNMR ......................................................... 68

6.3 Isothermal crystallization as visualized by PLM .................................................................... 70

6.3.1 Start of isothermal crystallization at 20°C ..................................................................... 70

6.3.2 6 week follow-up ........................................................................................................... 71

General conclusions .............................................................................................................................. 74

Further research ................................................................................................................................... 76

References............................................................................................................................................. 77

Appendix I: RSM results ........................................................................................................................ 85

Appendix II: Isothermal crystallization after 1 min ............................................................................... 86

Appendix III: Isothermal crystallization after 1 week, 3 and 5 weeks .................................................. 87

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis VI

List of abbreviations

A Arachidic acid

ACN Acetonitrile

AV Acid value

CB Cocoa butter

CBA(s) Cocoa butter alternative(s)

CBE(s) Cocoa butter equivalent(s)

CBEX Cocoa butter extender

CBI Cocoa butter improver

CBR(s) Cocoa butter replacer(s)

DAG(s) Diacylglycerol(s)

DCM Dichloromethane

DSC Differential scanning calorimetry

ELSD Evaporative light-scattering detector

FAM Fatty acid mixture

FFA(s) Free fatty acid(s)

GC Gas chromatography

HMFS Human milk fat substitutes

HOSO High oleic sunflower oil

HPLC High performance liquid chromatography

L Linoleic acid

LFA(s) Long chain fatty acid(s)

MAG(s) Monoacylglycerol(s)

MFA(s) Medium chain fatty acid(s)

O Oleic acid

OF1 First Olein fraction

OF2 Second Olein fraction

OOO Triolein

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis VII

P Palmitic acid

p-AV p-anisidine value

PLM polarized light microscope

PMF Palm mid fraction

pNMR Pulsed nuclear magnetic resonance

POP 1,3-dipalmitoyl-2-oleoyl-glycerol

POSt 1(3)-palmitoyl-3(1)stearoyl-2-oleoyl-glycerol

PV Peroxide value

RM IM Rhizomucor miehei immobilized

RSM Response surface methodology

PUFA(s) Polyunsaturated fatty acid(s)

St Stearic acid

SF1 First Stearin fraction

SF2 Second Stearin fraction

SFC Solid fat content

StOSt 1,3-distearoyl-2-oleoyl-glycerol

SPD Short path distillation

SSS Trisaturated TAG

SST(s) Specific-structured triacylglycerol(s)

SSU Saturated-saturated-unsaturated TAG

SUS Disaturated TAG

SUU Monosaturated TAG

TAG(s) Triacylglycerol(s)

UUU Tri-unsaturated TAG

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis VIII

List of figures Figure 1: Polymorphic transitions of CB (Van Malssen et al., 1999). ...................................................... 5

Figure 2: Steps of the enzymatic hydrolysis of fats and oils (Xu, 2003). ............................................... 11

Figure 3: The enzymatic esterification (Xu, 2003). ................................................................................ 11

Figure 4: The enzymatic alcoholysis (Xu, 2003). .................................................................................... 12

Figure 5: The enzymatic acidolysis between a TAG (XXX) and a FFA (Y) (Xu, 2003). ............................ 12

Figure 6: The main reactions and side reactions of the enzymatic acidolysis for a TAG (LLL) and a FFA

(M and L) using a sn-1,3 specific lipase (Xu, 2003). ............................................................................ 13

Figure 7: The ester-ester exchange reaction between two TAGs (XXX and YYY) with the help of a sn-

1,3 lipase. X and Y are two types of fatty acids (Xu, 2003). ................................................................ 13

Figure 8: Process scheme for SPD (Xu et al., 2002). .............................................................................. 17

Figure 9: Enzymatic acidolysis reaction on a big scale in optimized conditions. .................................. 22

Figure 10: SPD equipment (Oleon, Belgium). ........................................................................................ 22

Figure 11: Non-isothermal DSC results of HOSO. .................................................................................. 29

Figure 12: Process scheme to find the optimum conditions for the enzymatic acidolysis reaction. .... 30

Figure 13: The % TAG (POP, POSt and StOSt) and trisaturated TAGs (SSS) at different sampling times.

............................................................................................................................................................... 32

Figure 14: The % TAG (POP, POSt and StOSt) using different temperatures. ....................................... 33

Figure 15: The % TAG (POP, POSt and StOSt) and the amount of MAG + DAG formed, using different

water contents. ................................................................................................................................... 34

Figure 16: The % TAG (POP, POSt and StOSt) using different enzyme loads. ....................................... 35

Figure 17: The percentage of the TAG (POP, POSt and StOSt) for different substrate ratios. .............. 36

Figure 18: The percentage of FFA, DAG and TAGs (A) and a detail of the desired TAGs (POP, POSt and

StOSt) (B) for different ratios of glycerol added to HOSO. ................................................................ 37

Figure 19: The percentage of FFA (A) and DAG (B) for different ratios of glycerol and non-specific

enzyme added to HOSO. ..................................................................................................................... 39

Figure 20: The percentage TAG (POP, POSt and StOSt) for different ratios of glycerol and non-specific

enzyme. Method A (A), method B (B) and method C (C).................................................................... 40

Figure 21: Perturbation plot of SUS (left) and SUU (right) with A: substrate ratio (7 mol); B: enzyme

load (10%); C: water content (2%); D: temperature (65°C) and E: reaction time (6h). ...................... 43

Figure 22: Contour plot of the interaction between enzyme and temperature on % SUS (left) and %

SUU (right). ......................................................................................................................................... 44

Figure 23: Contour plot of the interaction between water and temperature on % SUS (left) and % SUU

(right). ................................................................................................................................................. 44

Figure 24: Contour plot for the optimal factor levels of % SUS (left) and % SUU (right). ..................... 45

Figure 25: Scheme of the obtained fractions after fractionation using method A or B. ....................... 47

Figure 26: Percentage of SSS TAGs in SF1 (A), UUU and SUU TAGs in OF2 (B) and SUS, UUU and SUU

TAGs in SF2 (C) compared between two fractionation methods. ...................................................... 48

Figure 27: Non-isothermal crystallization and melting profile of the interesterified product (product)

and the purified product (after SPD) as measured by DSC. ................................................................ 51

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis IX

Figure 28: Non-isothermal crystallization and melting profile of the fractions after fractionation

method A as measured by DSC. .......................................................................................................... 51

Figure 29: Non-isothermal crystallization and melting profile of the SF2 fractions after fractionation

method A and B as measured by DSC. ................................................................................................ 52

Figure 30: Non-isothermal (non-tempered and tempered) SFC curve of the product, purified product,

SF2 (CBE) and CB as measured by pNMR. .......................................................................................... 53

Figure 31: Percentage of POP, POSt and StOSt (A) and SUU, SSS (B) TAGs in the CB/ CBE mixtures. .. 55

Figure 32: POP/POSt/StOSt ternary diagram showing the position of CB, vegetable fats used as CBE

and the enzymatically produced CBE (Padley et al., 1981; Smith, 2001). .......................................... 56

Figure 33: Percentage of SSS, SUS, SSU and SUU TAGs in the CB/ CBE mixtures; results obtained by

silver ion HPLC. ................................................................................................................................... 57

Figure 34: Non-isothermal crystallization and melting profile of CB and the mixtures with CBE as

measured by DSC. ............................................................................................................................... 58

Figure 35: Non-isothermal non-tempered (A) and tempered (B) SFC curve of the CBE-CB mixtures as

measured by pNMR. ........................................................................................................................... 60

Figure 36: Non-isothermal SFC curve: comparison of tempered and non-tempered CB and pure CBE

as measured by pNMR. ....................................................................................................................... 61

Figure 37: SFC melting curves indicating the hardness (A), heat resistance (B) and waxiness (C) of CB

and mixtures with CBE (Depoortere, 2011). ....................................................................................... 62

Figure 38: Isothermal diagram of the mixtures of CBE and CB. ............................................................ 63

Figure 39: Isothermal crystallization of CB at 20°C as measured by DSC. ............................................. 64

Figure 40: Isothermal crystallization at 20°C of the different ratios of CB and CBE. ............................ 65

Figure 41: Influence of Sat FA to Unsat FA and SUS to SUU TAGs ratios on aF and tind. ........................ 67

Figure 42: Isothermal crystallization at 20°C of CB and mixtures with CBE as measured by pNMR. ... 69

Figure 43: Isothermal crystallization at 20°C after 60 min as visualized by PLM: 0 (a), 20 (b), 40 (c), 60

(d), 80 (e) and 100% (f) CBE. ............................................................................................................... 71

Figure 44: Isothermal crystallization at 20°C as visualized by PLM for CB, 20, 40, 60, 80 and 100% CBE.

The microstructure is given after 24h, 2 weeks, 4 weeks and 6weeks. ............................................. 73

Figure 45: Isothermal crystallization at 20°C after 1 min as visualized by PLM: 0 (a), 20 (b), 40 (c), 60

(d), 80 (e) and 100% (f) CBE. ............................................................................................................... 86

Figure 46: Isothermal crystallization at 20°C as visualized by PLM for CB, 20, 40, 60, 80 and 100% CBE.

The microstructure is given after 1 week, 3 and 5 weeks. ................................................................. 87

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis X

List of tables Table 1: Overview of the CBAs (Depoortere, 2011). ............................................................................... 7

Table 2: Vegetable fats allowed to use as CBE in chocolate according to EU Directive 2000/36/EC. .... 8

Table 3: An overview of the enzymatic interesterification in order to produce CBEs (Depoortere,

2011). .................................................................................................................................................. 15

Table 4: An overview of the tested enzymatic acidolysis parameters and their range. ....................... 21

Table 5: Distillation parameters in SPD. ................................................................................................ 23

Table 6: Results of the quality tests and the FFA composition of HOSO. ............................................. 27

Table 7: TAG composition of HOSO with P: palmitic acid, St: stearic acid, O: oleic acid and L: linoleic

acid. ..................................................................................................................................................... 28

Table 8: Three different methods in which glycerol and Novozyme 435 were added to the acidolysis

reaction. .............................................................................................................................................. 38

Table 9: The five factors used for RSM with the unit and lower and upper limit. ................................ 41

Table 10: Regression coefficients of the quadratic model for the response variables. ........................ 42

Table 11: Analysis of variance (ANOVA) for the response surface quadratic model. ........................... 42

Table 12: Optimum conditions for each combination of parameters and predicted amounts of SUS

and SUU given by RSM. ....................................................................................................................... 45

Table 13: Model verification. ................................................................................................................ 45

Table 14: TAG composition of the product after SPD. .......................................................................... 46

Table 15: Parameters Tonset (°C), Tpeak (°C), meting heat (J/g) and width at half height (°C) of DSC

melting profile (non-isothermal). ....................................................................................................... 50

Table 16: FA composition of CB and CBE. The results are the average of two repetitions. .................. 54

Table 17: Parameters Tonset (°C), Tpeak (°C), melting heat (J/g) and width at half height (°C) of DSC

melting profile (non-isothermal) for mixtures of CB and CBE. ........................................................... 58

Table 18: Parameters aF (J/g), tind (h) and K (h-1) of the Foubert model for mixtures of CB and CBE. .. 67

Table 19: The level of the factors, and the amount of % SUS and % SUU formed. .............................. 85

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis XI

Samenvatting Cacaoboter (CB) is het meest optimale ingrediënt om de specifieke en gewenste eigenschappen van

chocolade te verkrijgen. Door de stijgende prijzen en onzekerheid in aanbod, is de industrie

genoodzaakt te zoeken naar alternatieven voor CB. Het doel van dit onderzoek was om een

cacaoboter equivalent (CBE) te produceren uit ‘High Oleic Sunflower Oil (HOSO)’ met behulp van

enzymatische acidolyse. Om de triacylglycerol (TAG) samenstelling van HOSO gelijkaardig aan die van

CB te maken, werd palmitine (P), stearine (S) zuur en geïmmobiliseerd lipase van Rhizomucor miehei

(RM IM) gebruikt. Dit lipase beïnvloedt selectief de vetzuren op de sn-1- en sn-3- positie van de TAGs.

Het eerste deel van dit onderzoek bestond erin de goede kwaliteit van HOSO en de vereiste

eigenschappen die nodig zijn om het als bron voor enzymatische acidolyse te gebruiken, te

bevestigen. Het vetzuurprofiel gaf aan dat er 84% oleïne zuur (O) en 62% trioleïne (OOO) aanwezig

was. De hoeveelheid vrije vetzuren (FFA), peroxide getal (PV), para-anisidine (p-AV) en totox getal

voldeden allen aan de specificaties vereist voor voedingsmiddelen.

Als tweede werden de parameters van de acidolyse reactie geoptimaliseerd met behulp van

‘response surface methodology (RSM)’. Dit resulteerde in een reactietijd van 8u, reactietemperatuur

van 65°C, 1% water, 8.54% enzym en een substraat ratio van 7.99:1 (mol vetzuren: mol HOSO). Een

aantal pogingen werden ondernomen om de opbrengst van de reactie te verhogen door glycerol en

niet-specifiek enzym toe te voegen. Het resultaat was een afname van FFA van 65% tot 9%. De

hoeveelheid diglyceriden nam toe en de hoeveelheid gewenste TAGs (POP, POSt en StOSt) nam af.

In het derde deel werd het gehalte FFA in het product teruggebracht tot 0.32% door middel van

‘short path distillation (SPD)’. Het gehalte mono-verzadigde (SUU) en volledig verzadigde (SSS) TAGs

werd gereduceerd door fractionatie. De bekomen CBE bestond uit 8.42% SUU en 4.61% SSS TAGs in

vergelijking met 1.64% SUU en 1.56% SSS in CB. In vergelijking met CB, had het een gelijkaardige

hoeveelheid POP en lagere hoeveelheden aan POSt en StOSt.

Tenslotte werd de CBE in verschillende ratio’s gemengd met CB. De analyse van de mengsels met

differentiële scanning calorimetrie (DSC) en gepolariseerde licht microscopie (PLM), toonde aan dat

een hoger gehalte aan CBE resulteerde in een trage kristallisatie. Dit is te wijten aan het hoger

gehalte SUU TAGs in CBE. Met behulp van ‘pulsed nuclear magnetic resonance (pNMR)’, werd

aangetoond dat het gehalte SUU en SSS TAGs resulteerde in een lager gehalte vast vet (SFC) bij lage

en een hoger SFC bij hogere temperaturen in vergelijking met CB. Door gebruik te maken van

enzymatische acidolyse, was het mogelijk een CBE te produceren waarvan de chemische

samenstelling die van CB sterk benaderde. De fysische eigenschappen van de CBE weken significant

af van die van CB wat resulteerde in een heel traag kristallisatieproces.

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis XII

Summary Cocoa butter (CB) is the best ingredient to obtain the specific and desired characteristics of chocolate.

Due to the increase in price and uncertainty in supply, industry is forced to seek alternatives to CB.

The aim of this research was to produce a cocoa butter equivalent (CBE) by enzymatic acidolysis

starting from High Oleic Sunflower Oil (HOSO). To make the triacylglycerol (TAG) composition of

HOSO closer to that of CB, palmitic (P), stearic (St) acid and immobilized lipase from Rhizomucor

miehei (RM IM), which selectively affected the fatty acid (FA) in the sn-1- and sn-3-position of the

TAGs, were used.

In the first part of the research, the good quality of HOSO and the required characteristics to be used

as a source for the enzymatic acidolysis reaction were confirmed. The FA profile showed 84% oleic

acid (O), and 62% of its TAGs was triolein (OOO). The amount of free fatty acids (FFA), peroxide value

(PV), para-anisidine (p-AV) and totox value all complied with the regulations set for food.

Secondly, the parameters of the acidolysis reaction were optimized by response surface

methodology (RSM) resulting in a reaction time of 8h, 65°C as reaction temperature, 1% of water, an

enzyme load of 8.54% and a substrate ratio of 7.99 mol fatty acids (FA) for 1 mol HOSO. Some trials

were performed in order to improve the yield by adding glycerol and non-specific enzyme. The result

was a reduction of FFA from 65% to 9%. However, the amount of diglycerides (DAGs) increased a lot

and the amount of desired TAGs (POP, POSt and StOSt) decreased.

In the third part, the product was purified by short path distillation (SPD) to reduce the amount of

FFA to 0.32%. Fractionation was applied to reduce the amount of saturated-unsaturated-unsaturated

(SUU) and trisaturated (SSS) TAGs. The resulted CBE contained 8.42% SUU and 4.61% SSS TAGs

compared to 1.64% SUU and 1.56% SSS in CB. The CBE had a similar amount of POP, and lower

amounts of POSt and StOSt than CB.

Finally, the CBE was mixed in different ratios with CB. The results, as measured by differential

scanning calorimetry (DSC) and polarized light microscopy (PLM), showed that a higher amount of

CBE resulted in a slow crystallization. This was due to the high amount of SUU TAGs present in the

CBE. With pulsed nuclear magnetic resonance (pNMR), it was shown that the higher amount of SUU

and SSS TAGs resulted into a lower solid fat content (SFC) at low temperatures and an increased SFC

at high temperatures compared to CB.

With the enzymatic acidolysis, it was possible to produce a CBE with a chemical composition that was

very close to that of CB. However, the physical characteristics of the CBE differed significantly by

means of a very slow crystallization process compared to CB.

Production of Cocoa Butter Equivalent through Enzymatic Acidolysis 1

Introduction CB is the most important ingredient of chocolate. However, the supply and price of CB can be

uncertain and variable. The price of CB in January 2005 was 1549 US $ per ton while in March 2012

the price was 2359 US $ per ton (ICCO, 2012). For this reason, a lot of research has been done in

order to find cheaper and better available alternatives. The most used alternatives are the CBEs

which are chemically and physically similar to CB. However, the use of these CBEs in the European

Union is very regulated by the EU Directive 2000/36/EC.

CBEs are non-lauric vegetable fats which are completely compatible with CB. Therefore, the CBE can

replace the expensive CB in chocolate. Although the EU Directive 2000/36/EC states that CBEs

produced by enzymatic means or from a source other than the 6 permitted oils, is not allowed to use

in chocolate, a lot of research is performed to use cheap, commercial available oils and enzymatic

esterification. An example of such a natural oil is HOSO which is rich in O (Xu X., 2000).

The goal of this research was to produce a CBE by enzymatic acidolysis of the cheap and commercial

available HOSO with a fatty acid mixture (FAM), mainly consisting of P and St.

In the first part of the research, the characterization of the starting oil and the evaluation of its

quality was discussed. The chemical characterization consisted of analyzing the FA and TAG

composition. To evaluate the oxidative quality of HOSO; the amount of FFA, PV, p-AV and totox value

were determined. Also, the crystallization and melting behavior were measured by DSC and pNMR.

In the second part, the actual parameters of the enzymatic acidolysis reaction such as reaction time,

reaction temperature, water content, enzyme load and substrate ratio were optimized on a small

scale and later by RSM in order to obtain the highest yield possible of POP, POSt and StOSt. Also,

some trials were done by using glycerol and non-specific enzyme in order to increase the product

yield. This was followed by the purification of the interesterified product by SPD and fractionation to

produce a pure CBE.

In the following part, the physico-chemical characterization of the produced CBE was studied. TAG

composition by High Performance Liquid Chromatography (HPLC) and FA profile by Gas

Chromatography (GC) were determined. Melting and crystallization properties were analyzed by DSC

and pNMR.

Finally, the produced CBE was mixed in different ratios with CB in order to analyze the compatibility

with CB. PLM was applied to analyze the crystal microstructure in these mixtures over a period of 6

weeks.


Literature

1. Modification of fats and oils

Fats and oils play an important role in many food products that are consumed every day. In fact, it is

important for the texture, aroma and mouth sensations of the products (Wassell et al., 2010).

When modifying fats, not only the physico-chemical properties such as solid fat content (SFC) and

melting profile are changed, but also the altered functional role of the fat in the final application

cannot be neglected. The composition, polymorphism, SFC and the microstructure of triacylglycerols

(TAGs) are very important in the structure of lipid-based products. Structuring TAGs results in a

bigger diversity of the molecules and their configurations compared to the original fat; in that way,

the right physical properties can be acquired for the final application (Wassell et al., 2010).

Modifying fats and oils can be done chemically and also with the use of enzymes. During the last

years, the enzymatic methods became more important and popular; therefore lots of the chemical

methods can be replaced by enzymatic ones. When using enzymes, the reactions are more specific

and the most crucial advantage is that there is no need for chemical reagents. The enzymatic

modification can be done through hydrolysis, esterification, acidolysis, alcoholysis and ester-ester

exchange. With the help of enzymes, human milk fat substitutes (HMFS), cocoa butter equivalents

(CBE), confectionary anti-bloom agents, diacylglycerol (DAG) cooking oil, polyunsaturated fatty acid

concentrates, etc. are produced (Xu et al., 2006).

TAGs, which are modified, are called structured TAGs. Nowadays, many nutritional and functional

specific-structured triacylgycerols (SSTs) are produced. The nutritional and functional properties are

due not only to the fatty acid profile, but also to the fatty acid distribution on the glycerol backbone.

The specific characteristics of breast milk fat and cocoa butter (CB) are due to the fatty acid

distribution on the glycerol backbone. Because of the high specificity that is required to produce the

SSTs, it is impossible to apply chemical methods and only lipases with a high regiospecificity are used

(Xu, 2000).

Fractionation, hydrogenation, and interesterification are three different methods that are used to

modify fats and oils. Also blending different types of oils is a way to obtain a product with desired

characteristics.


1.1 Fractionation

Fractionation is the method of physically separating high melting point fractions (stearin) from low

melting point ones (olein). Fractionation is a thermo-mechanical separation process and is

completely reversible (Kellens et al., 2007). Palm oil is the most common oil which is fractionated

quite often. Normally, palm oil can be fractionated into palm olein, palm stearin, palm mid fraction

(PMF) and so on. The separation is based on the difference in melting points of the fractions (Xu,

2000).

In the first step of the fractionation process, the fat sample should be melted completely, followed

by a slow cooling which is very gentle and without agitation in order to obtain large crystals. If a fast

cooling would be applied, it would result in small fat crystals. To obtain a good separation, the

crystals should be firm and of uniform size. During the cooling and crystallization, which is a two-step

process involving nucleation and crystal growth, the viscosity of the solution will increase (Kellens et

al., 2007). This crystallization is done in a controlled way so the fraction with the highest melting

point will crystallize first and the other fraction will still be liquid because of the lower melting point.

The liquid and solid fractions have different physical and chemical compositions and the two

fractions can be separated using filtration (Wassell et al., 2010).

There are three types of fractionation: dry fractionation, solvent fractionation and detergent

fractionation. Dry fractionation is the simplest, cheapest and a ‘green’ method because no solvents

are used and there are no losses of product. Viscosity limits the use of dry fractionation in a single

step because it restricts the degree of crystallization. That is why this method is mostly done in a

multi-step operation (Kellens et al., 2007). Solvent fractionation requires solvent and in most cases;

hexane, acetone or 2-nitro-propane is used (Bockisch, 1998). This type of fractionation is more

efficient but has higher operating costs. The other disadvantage of this method is the oil entrained

between the crystals. This can be removed by centrifugation, vacuum filtration or pressing (Salas et

al., 2011).On the other hand, the advantages of using solvents are a faster nucleation and growth of

the crystals, a lower viscosity which leads to easier filtration, a dilution of the fat that makes the heat

transfer easier and the possibility to wash the crystals repeatedly with the solvent to reduce the

amount of entrained oil (Timms, 2005). The first step in detergent fractionation, is the fractional

crystallization followed by adding water containing an aqueous detergent such as sodium lauryl

sulphate and an electrolyte (magnesium sulphate or sodium sulphate). The crystals become

dispersed in the solution and the electrolyte facilitates the agglomeration of the oil droplets during

mixing. Finally, the crystal separation is completed by centrifugation (Rajah, 1996).


1.2 Hydrogenation

To get a specific and desired melting behavior of fat blends, hydrogenation or partial hydrogenation

processes can be used. This technique is mostly applied to give firmness to margarines, plasticity and

emulsion stability to shortenings (Wassell & Young, 2007). Unsaturated fatty acids have double

bounds and are usually in the cis configuration. Hydrogenation, which adds hydrogen atoms to the

double bounds, results in a higher degree of saturation and on top of that, a more rigid structure of

the TAG and a higher melting point are obtained. But the disadvantage of the hydrogenation is that

the cis isomers can be changed into the trans ones and these trans fatty acids have negative health

effects (Wassell et al., 2010).

1.3 Interesterification

In general, it is a process that results in the rearrangement of the distribution of the fatty acids on

the glycerol backbone. Interesterification is an alternative to hydrogenation but without the

formation of trans fatty acids (Wassell & Young, 2007). It can be done in a chemical or enzymatic way,

and within or between TAGs. There are different possibilities of enzymatic interesterifications:

alcoholysis, acidolysis and ester-ester exchange. The enzymes, which are used, can be specific or

non-specific (Wassell et al., 2010).

1.4 Blending

Another way to modify fats and oils is blending oils with fully hardened ones to obtain a product with

desired physical characteristics. Blending vegetable oils from different sources is an alternative for

the hydrogenation of vegetable oils but with the right physico-chemical properties and nutritional

requirements that are demanded. Another advantage of this technique is that there is no chemical

modification. However, a disadvantage is that the blending of the right amounts of oil is often a

process of trial and error (Wassell & Young, 2007).

2. Cocoa butter

Chocolate contains many ingredients of which CB is the most important. It is the most expensive

ingredient and one third of the cost of chocolate is due to the CB (Widlak, 1999).

Some of the unique characteristics of chocolate, for instance, the viscosity and the rheological

properties depend on the crystallization of the CB. Also, the snap and the surface gloss of the

chocolate are due to the TAG composition of the CB (Widlak, 1999). CB is responsible for the

brittleness at room temperature, the cooling effect in the mouth, and for the quick and complete

melting of the chocolate around body temperature (27-33°C). However, the TAG composition of CB

can vary depending on the geographical source.


2.1 Chemical properties

CB consists mainly of three fatty acids: palmitic acid (C16:0, 20 - 26%), stearic acid (C18:0, 29 - 38%),

and oleic acid (C18:1, 29 - 38%). Linoleic acid (L, C18:2, 2 - 4%) and arachidic acid (A, C20:0, 1%) are

also present in considerable amounts. More than 70% of its TAGs are symmetrical with oleic acid (O)

at the sn-2 position. The three most important TAGs are POP (21%), POSt (40%) and StOSt (27%). The

difference in amount of these fatty acids and TAGs are due to the origin of the CB (Xu, 2000; Talbot,

2009b; Smith, 2001).

2.2 Physical properties

Because of the composition of the TAGs in the CB, it can crystallize into 5 or 6 polymorphic forms,

depending on the reference. According to Van Malssen et al. (1999), when using the classification,

form V and VI are the most stable ones and these do not crystallize directly from the melt. Therefore,

a recrystallization from a metastable polymorphic form is necessary as presented in figure 1. The

desired polymorphic form of chocolate is the βV-polymorph. βVI is the most stable polymorphic form

which is typical for fat bloom (Timms, 2003). Also, the typical melting point of the CB depends on the

polymorphic form and this can range from 15 to 36°C (Huyghebaert, 1971).

Figure 1: Polymorphic transitions of CB (Van Malssen et al., 1999).


3. Cocoa butter alternatives

The discussed techniques to modify oils can also be used to produce alternatives to CB starting from

vegetable oils. As mentioned earlier, CB is an expensive ingredient and prices can be unpredictable,

also the supplies can be uncertain. Thus, for economical and technical reasons, producers are forced

to seek alternatives to replace the CB (Xu, 2000).

Vegetable fats can be used as alternatives to CB in chocolate. These replacer fats are called cocoa

butter alternatives (CBA). CBA can be divided into three categories: cocoa butter equivalents (CBE),

cocoa butter substitutes (CBS), and cocoa butter replacers (CBR). The CBAs are mostly mixtures of

different vegetable fats. The CBEs are non-lauric fats with similar physical and chemical properties as

CB. They can be mixed with the CB without changing the physical properties of it. The CBR are also

non-lauric fats with a similar fatty acid distribution but a completely different structure of TAGs to CB.

Finally, the CBS are lauric plant fats that are chemically totally different to CB but with some physical

similarities. The CBS and CBR are found in compound chocolate of which the fat phase contains other

fats than real CB, for instance in chocolate-coatings and ice-cream (Lipp & Anklam, 1998; Smith, 2001;

Stewart & Timms, 2002).

CBA should be compatible with CB in brittleness and melting behavior. The melting and

crystallization characteristics are mainly due to the TAG composition. Thus, if a substitute of CB is

produced, several aspects such as the melting behavior will be crucial. The melting behavior has to

be very similar to that of CB in order to achieve the same mouth feeling and the addition of the CBA

should not change the crystallization of the CB (Lipp & Anklam, 1998). Table 1 gives an overview of

the classification of CBAs.


Table 1: Overview of the CBAs (Depoortere, 2011).

CBE CBR CBS

Origin

Illipé butter

Palm oil

Sal fat

Shea butter

Kokum butter

Mango kernel fat

Palm oil

Soybean oil

Rapeseed oil

Cottonseed oil

Palm kernel oil

Coconut oil

Processing

Hydrogenation

Fractionation

Interesterification

Hydrogenation

Fractionation

Hydrogenation

Fractionation

Interesterification

TAG composition Similar to CB

Different from CB Different from CB

Lauric acid Non lauric

Non lauric Lauric

(45-55% lauric acid)

Compatibility to CB Compatible Compatible in small

ratios

Incompatible

Crystallization

Tempering to obtain

stable polymorphic

form

Crystallize directly

from the melt in the

stable polymorphic

form

Crystallize directly

from the melt in the

stable polymorphic

form

Application

5% replacement on

total product

Compound

Compound

Compound

Remark

Cocoa butter extender

(CBEX)

Cocoa butter improver

(CBI)

High level of trans fatty

acids


3.1 Cocoa butter equivalents

One type of CBAs are CBEs which are totally compatible with CB and, for this reason, it can be mixed

with the CB without any problem. Therefore, a lot of research is done for CBEs because they have the

closest characteristics to CB (Xu, 2000). The CBEs can be divided into two groups: the cocoa butter

extenders (CBEX) and the cocoa butter improvers (CBI). CBEX are mixable with CB but not in every

ratio. The CBI have a high content of StOSt and this will increase the SFC. Due to the higher amount

of solid fat, the melting point and the hardness is increased. Chocolate with CBI has a better

resistance to softness and formation of fat bloom at higher ambient temperatures; for example in

summer or in tropical regions (Timms, 2003).

3.1.1 Legislation

The European Union has established the EU Directive 2000/36/EC relating to cocoa and chocolate

products intended for human consumption. Up to now, only 5% vegetable fats other than CB are

allowed in chocolate in some of the Member States of the European Union. These vegetable fats

should be CBEs and therefore be defined according to the technical and scientific criteria and meet

the following criteria before they can be used in chocolate (EU Directive 2000/36/EC).

They are non-lauric vegetable fats, which are rich in symmetrical monounsaturated TAGs of

the type POP, POSt and StOSt.

They are miscible in any proportion with CB and are compatible with its physical properties

(melting point and crystallization temperature, melting rate, need for tempering phase).

They are obtained only by the processes of refining and or fractionation which excludes

enzymatic modification of the TAG structure.

Table 2 gives the 6 vegetable fats that are allowed to use as a CBE in chocolate according to the EU

Directive.

Table 2: Vegetable fats allowed to use as CBE in chocolate according to EU Directive 2000/36/EC.

Name of the vegetable fat Scientific name of the plants from which the fat

can be obtained

Illipé, Borneo tallow or Tengkawang Shorea spp.

Palm oil Elaeis guineesis, Elaeis olifera

Sal Shorea robusta

Shea Butyrospermum parkii

Kokum gurgi Garcinia indica

Mango kernel Mangifera indica

As an exception, coconut oil can be used in chocolate but only for the manufacture of ice cream and

similar frozen products.


If the previously mentioned vegetable fats are used in chocolate products, the consumer should be

informed correctly and objectively. They should be mentioned in the list of ingredients and the

product should be labeled with: ‘contains vegetable fats in addition to cocoa butter’ (EU Directive

2000/36/EC).

Countries outside the EU have their own regulations and these can differ from the European

legislation. For instance, it is not permitted in the United States to use vegetable fats other than CB in

chocolate, but the American legislation allows the use of it in chocolate coatings and vegetable fat

coatings. There are also countries where more than 5% of vegetable fats can be used in chocolate

but the products cannot be labeled as ‘chocolate’. (Talbot, 2009b)

3.1.2 Sources

The fats that can be used to produce CBEs are mentioned in table 2 and these are palm oil, illipé,

shea and also sal, kokum gurgi and mango kernel. In other words, these are the types of fats that are

allowed by the EU.

3.1.2.1 Palm oil

Palm oil is obtained from the flesh of the fruit of Elaeis guineensis and it is mostly produced from

trees in Malaysia or Indonesia. The fatty acid composition of palm oil and PMFs can be typical for a

specific region but it mostly consists of P and O. To make the fatty acid composition closer to CB, the

PMF can be interesterified with P or St. Palm oil can be fractionated in palm olein and palm stearin.

In addition, the content of the different TAGs depends strongly on the fraction and the used

technique to obtain that fraction but in general mainly POP and POO are found (Lipp & Anklam,

1998).

3.1.2.2 Illipé butter

Other names for Illipé fat are Borneo tallow, engkabang or tenkgawang. The fat is obtained from the

seed kernels of the Shorea stenoptera, this tree grows in Borneo, Java, Malaysia and the Philippines.

The fatty acid composition of the fat resembles somewhat CB because of the high St content. The

amount of O and St in the illipé fat is more or less equal followed by P. It has a relatively high level of

POSt and StOSt. Before using this fat, it needs to be refined (Lipp & Anklam, 1998; Storgaard, 2000).


3.1.2.3 Kokum butter

Kokum butter is also called Goa butter, it is obtained from the seed kernels of the Garcinia indica or

the Kokum tree. This is an evergreen tree that grows in the tropical forests of India. Solvent

extraction is mostly used to obtain the oil from the seeds. The butter consists of high amounts of St,

followed by O (Lipp & Anklam, 1998). When kokum is interesterified with P and/or St, it was claimed

to resemble CB very well in both the fatty acid and TAG composition, as well as the melting behavior

(Sridhar et al., 1991). The TAG composition of kokum butter consists mostly of StOSt (77%), StOO

(12%) and POSt (8%). Before using it, the fat has to be refined (Sridhar & Lakshminarayana, 1991).

3.1.2.4 Mango kernel fat

This fat is obtained from the seed kernels of the fruit of the mango tree or Mangifera indica. Solvent

extraction is necessary to release the fat because only 6 to 15% fat is present in the kernel. The most

common TAG is StOSt which accounts for 40.6% of the TAG content. To obtain higher levels of StOSt,

solvent fractionation is used and after this, a refining process of the fat is needed (Timms, 2003).

3.1.2.5 Sal fat

Other names for Sal fat are Borneo tallow or tenkgawang tallow. It is obtained from the seed kernels

of Shorea robusta which grows in Borneo, Java, Malaysia and the Philippines. In older references, this

fat is often confused with Illipé. The fatty acid composition has some resemblance to CB because of

the high amount of St. It is also high in O, followed by P. These fats resemble CB very closely due to

their fatty acid composition, of which is about 33% O, the same amount of St and about 24% P. On

top of that, Sal fat contains a considerable amount of A. This makes the most common TAGs in this

fat; StOSt (42-52%) and StOA (20%). Also Sal fat needs to be refined and fractionated before using it

as a CBE (Lipp & Anklam, 1998).

3.1.2.6 Shea butter

Shea butter is also called Karite butter or Galam butter. It is obtained from the nuts of the tree

Butyrospermum parkii which is mainly found in West Africa. The geographical origin has a big

influence on the fatty acid composition of this fat but it mainly consists of O and St. The main TAG in

Shea is StOSt, so after fractionation, this fraction is mostly used to produce CBEs. Next to this

fractionation step, refining needs to be done before it can be used as a CBE (Lipp & Anklam, 1998).


3.1.3 Production

Nowadays, for the modification of lipids in order to produce CBEs, enzymes are more used than

chemical methods. A liquid enzyme can be used or an immobilized enzyme (the enzyme is coated in a

monolayer on a solid particle). According to the legislation, the use of enzymatic produced CBEs is

not allowed within the European Union, nevertheless, a lot of research now is based on the use of

enzymes. Such enzymes or hydrolases, need a lot of water in the system for the hydrolysis of the

fatty acids from the TAGs. But in an environment with only a very small amount of water, these

enzymes can also be used to catalyze the reverse reaction; this is the so called esterification. Next to

the esterification, other reactions, called the interesterification reactions, are used to produce the

CBEs. The interesterification is the reaction between an ester and a fatty acid, an alcohol or another

ester. Different interesterification techniques are alcoholysis, acidolysis and ester-ester exchange.

The enzymatic interesterification reaction is a two step mechanism: hydrolysis and esterification

(Rozendaal & Macrae, 1997).

3.1.3.1 Hydrolysis

In nature, enzymes perform hydrolysis; this means they convert TAGs into DAGs and in the final step,

monoacylglycerols (MAGs) and free fatty acids (FFA) are formed. Thus, the hydrolysis of oils and fats

involves three steps from TAG to glycerol and FFA. In figure 2, this process is given schematically (Xu,

2003).

Figure 2: Steps of the enzymatic hydrolysis of fats and oils (Xu, 2003).

3.1.3.2 Esterification

This is the inverse reaction of hydrolysis and is only possible in an environment with a very small

amount of water, a so called micro-aqueous reaction system. In this system, the hydrolysis is

minimized while in water abundant systems, the hydrolysis is the main reaction. Esterification is

actually the simple reaction between an organic acid and an alcohol. It is the condensation of FFA on

the glycerol backbone. As can be seen in figure 3, water is one of the products that are formed during

the reaction. Therefore, it is very important to remove the water to shift the thermodynamic

equilibrium to the synthesis. The reaction can be carried out in systems using solvents or in solvent-

free systems (Rodrigues & Fernandez-Lafuente, 2010).

Figure 3: The enzymatic esterification (Xu, 2003).


The water, which is produced during the reaction, can shift the reaction equilibrium towards

hydrolysis if it is not continuously removed. On the other hand, water cannot be entirely removed

because a certain amount of water is necessary to maintain a high enzyme activity, this requires a

higher aw range while a high product yield requires aw as low as possible. One of the options to

remove water from the system is bubbling dry air or nitrogen gas in the reactor (Oh et al., 2009).

Another possibility are molecular sieves.

3.1.3.3 Alcoholysis

The alcoholysis technique is also performed using enzymes. It is the reaction between an ester and

an alcohol. Chemical alcoholysis is used in industry to produce MAG, DAG and biodiesels. Using

enzymes gives several advantages because of their high specificity. The reaction is schematically

shown in figure 4. The ester can be acylglycerols or TAGs, and the alcohol can be glycerol, methanol

or ethanol (Xu, 2003).

Figure 4: The enzymatic alcoholysis (Xu, 2003).

3.1.3.4 Enzymatic acidolysis

Another option of the interesterification method to modify fats and oils, is the enzymatic acidolysis.

This reaction involves an ester and an acid, the acid will be exchanged with another acid in the ester.

The enzymatic acidolysis is widely used for the production of CBEs. The reactions are catalyzed by sn-

1,3 specific lipases because the positional specificity is essential for the final product. This is clearly

shown in figure 5. In this figure, a sn-1,3 specific lipase is used, this means that the lipase will only

change the fatty acids on the first and third position of the TAG, in other words, the FFA (Y) will only

be implemented on position 1 and/or 3 (Esteban et al., 2011).

Figure 5: The enzymatic acidolysis between a TAG (XXX) and a FFA (Y) (Xu, 2003).

The used ester doesn’t always have to be a TAG as shown in figure 5, also DAG and MAG can be used

as ester.

A disadvantage is that the DAGs, which are formed during the reaction, can cause side reactions and

produce some by-products (Pacheco et al., 2010). Figure 6 gives an example of this problem.


Figure 6: The main reactions and side reactions of the enzymatic acidolysis for a TAG (LLL) and a FFA (M and L) using a sn-1,3 specific lipase (Xu, 2003).

Several factors affect the formation of by-products and acyl migration during the reaction. These

factors are an increase in temperature, reaction time, water content and water activity. There is also

a positive correlation found between the enzyme content and the acyl migration because the carriers

of immobilized lipases induce acyl migration. This carrier can be a resin or silica (Hoy & Xu, 2001).

Acyl migration is a disadvantage when CBEs are produced, it is not wanted that the oleic acid on the

sn-2 position shifts to another position on the glycerol backbone (Rodrigues & Fernandez-Lafuente,

2010). To reduce the acyl migration, one option is to use packed enzyme bed reactors instead of the

stirred tank reactors.

3.1.3.5 Ester-ester exchange

In the product, it is also possible to have an ester-ester exchange between two TAGs. Again, a sn-1,3

specific lipase is used and the fatty acids on the positions 1 and 3 will be exchanged. This reaction is

schematically presented in figure 7 (Xu, 2003).

Figure 7: The ester-ester exchange reaction between two TAGs (XXX and YYY) with the help of a sn-1,3 lipase. X and Y are two types of fatty acids (Xu, 2003).


4. CBE production

The use of lipases has several advantages over chemical catalysts. One of those advantages is that

the enzymes produce less by-products. Other advantages are lower energy consumption and better

product control. However, one of the major benefits of using lipase for the production of CBEs is the

regiospecificity of the enzymes. Enzymes have a high specificity but this can be affected by the pH,

temperature, concentration and the reaction medium.

Most enzymes that are used are microbial lipases. These are the most attractive ones for several

reasons; they are thermostable and don’t need a co-lipase or other different specifications (Xu, 2000).

Immobilized lipases are used in plenty of applications to improve the reusability of the very

expensive enzyme and to develop its stability and selectivity. Immobilization will also decrease the

potential inhibition of the used lipase. In fact, immobilization is mostly done by adsorption, it makes

the lipase also stable during the interesterification. This is because the lipase is not soluble in organic

solvents. For the immobilization, it is very important to choose the right support material as this can

affect the activity and the stability of the immobilized lipase (Wang et al., 2006). The immobilized

enzymes can be used at higher temperatures and especially in systems with very small amounts of

water (Chopra et al., 2008).

An overview of the research that has been performed on the production of CBEs using enzymatic

interesterification is illustrated in table 3.


Table 3: An overview of the enzymatic interesterification in order to produce CBEs (Depoortere, 2011).

Substrate Enzyme Reference

Mahau fat, kokum fat, dhupa fat,

sal fat, mango fat, fatty acid-

methyl ester

Lipozyme immobilized (IM) Sridhar et al., 1991; Xu, 2000

High oleic acid rapeseed oil Rhizopus arrhizus lipase,

lipozyme

Gitlesen et al., 1995

Palm oil, StStSt, stearic acid,

Stearic acid ethyl ester

Rhizomucor miehei lipase

Chinese vegetable tallow, fully

hydrogenated soybeen oil fatty

acids

Porcine pancreatic lipase Xu, 2000

Chinese vegetable tallow, stearic

acid

Porcine pancreatic lipase

PMF, stearic acid

Rhizopus arrhizus lipase

Teaseed oil, palmitic acid

Stearic acid

Porcine pancreatic lipase Xu, 2000; Wang et al., 2006

High oleate sunflower oil Lipozyme Smith, 2001

PMF and stearic acid Lipozyme Thermomyces

lanuginosis IM (TL IM)

Undurrage et al., 2001; Holm &

Cowan, 2008

Strychnos madagascariensis oil,

Trichelia emetic oil, Ximenia caffra

oil

Rhizomucor miehei lipase Khumalo et al., 2002

Refined bleached and deodorized

palm oil, fully hydrogenated

soybean oil

Lipozyme immobilized (IM) Abigor et al., 2003

Refined olive pomace oil

Porcine pancreatic lipase Ciftci et al., 2009

Palm oil

Carica papaya lipase Pinyaphong & Phutrakul, 2009

Pentadesma butyracea butter

Lipozyme TL IM Tchobo et al., 2009


The acidolysis reaction is performed using an enzyme that is sn-1,3 specific. One of the enzymes that

can be used is the lipase from Rhizomucor miehei (RM IM) or formerly known as Mucor miehei. This

enzyme is commercially available in the soluble and the immobilized form. Two important

characteristics of RM IM are the stability under diverse conditions, and the high activity. Thanks to all

of previous mentioned advantages, this enzyme has its main uses in fatty acids, oils and the

modification of fats as in the production of CBEs through enzymatic acidolysis (Rodrigues &

Fernandez-Lafuente, 2010).

RM IM is a very useful enzyme in systems where the water activity is held low. In an environment

with a low water activity, the enzyme performs active and stable; also the selectivity is greater at

lower aw (Rodrigues & Fernandez-Lafuente, 2010).

5. Product purification

The fat obtained after enzymatic acidolysis not only contains the desired SSTs but also more than 50%

consists of medium-chain (MFA) and long-chain fatty acids (LFA). Prior to the use of the product as

food, it is necessary to remove those FFA. Because of the high content of FFA, it is not easy to apply a

conventional distillation to remove them. To get rid of these FFA, short path distillation (SPD) can be

applied (Xu et al., 2002).

SPD is a thermal separation technique in which the boiling point of substances is lowered by using

high vacuum pressure. In this manner, the separation of heat-sensitive compounds, materials with a

low volatility and materials with a high molecular weight is possible (Lin & Yoo, 2009; Tovar et al.,

2011; Martins et al., 2006). Another name for SPD is molecular distillation, in which the distance

between evaporator and condensor is on the order of the average free path length of the molecules.

This system also operates under vacuum and it offers a very short residence time and a small hold up

volume (Tovar et al., 2011; Martins et al., 2006). It is a method that is frequently applied in lipid areas,

it has been used to purify MAGs, fraction PUFAs from fish oils, recover carotenoids from palm oil,

recover tocopherols, etc. (Xu et al., 2002). It is also often used to purify products that contain a lot of

MAGs and DAGs which have a strong effect on the crystallization behavior of fats (Lin & Yoo, 2009).

Important parameters that have to be considered and optimized are the temperature of the

evaporator, the feeding flow rate, the speed of the stirring roller and also the content of the FFAs;

since the method that leads to a lower FFA content will also results in a higher loss of tocopherols. An

important disadvantage of using too high temperatures, is that the amount of condensate in the

degasser pump will increase and this is not good for the performance of the distillation equipment.

High temperature has a negative effect on the amount of FFA so it is important to find the optimum

temperature (Xu et al., 2002; Martin et al., 2010).


The function of the roller in the system is to spread the feed equally on the inside surface of the

heating wall. In this way, the thickness of the film can be controlled. A faster roller speed will

improve the separation performance but more tocopherols and TAGs (due to splattering) will be lost

(Xu et al., 2002).

In the study of Xu et al. (2002), it was found that the flow rate has the most influence on the amount

of FFA and because it is strongly related to the heating capacity of the evaporator, makes it a very

essential parameter.

The process is schematically represented in figure 8. The distance between the evaporator and the

condensor is very short and a pressure drop is avoided.

The product after interesterification is brought into the feeding tank, the product goes to the

evaporator and is put as a thin layer on the inside of the wall by the stirring roller. The FFA are

evaporated and leave the equipment in the distillate receiver. A FFA trap with liquid nitrogen is

necessary to condense the FFA in order not to be sucked into the vacuum pump. The residue with

the desired part of the interesterification product is condensed and obtained by the residue receiver.

Figure 8: Process scheme for SPD (Xu et al., 2002).


6. Optimization of the reaction

The response surface methodology (RSM) is a statistical technique that is used in the investigation of

complex processes. There are only a reduced number of experiments necessary to provide enough

information to gain statistically acceptable results, which is the biggest advantage of RSM. It is a

method that is used a lot in food science research. Elibal et al. (2011) used RSM to optimize the

production of SSTs containing conjugated linolenic acid by enzymatic acidolysis. Melo Branco et al.

(2011) used RSM to model the production of SSTs from soybean oil after enzymatic acidolysis and

Shuang et al. (2009) optimized the production of SSTs by lipase-catalyzed acidolysis of soybean oil.

RSM is used to evaluate the effects of different variables in the process like reaction time, reaction

temperature, substrate ratio, enzyme load and water content, and it allows one to conclude which

variable will be the most vital (Shieh et al., 1995). It also enables the user to evaluate the effects on

the response variable(s) of multiple parameters in combination or alone. RSM can also predict the

behavior of the response variable(s) under given sets of conditions. Moreover, it is even possible to

find more than one optimum condition for the reaction due to different combinations of the

variables (Xu et al., 1998; Shekarchizadeh et al., 2009). When comparing RSM with classical one-

variable-at-a-time or full-factorial experiments, RSM performs faster and is less expensive (Shieh et

al., 1995).


Materials and methods

1. Substrates and enzyme

High Oleic Sunflower Oil (Radia 7363) and a mixture of free fatty acids (FAM) (RADIACID 0417) were

provided by Oleon company (Ertvelde, Belgium). The CB used as a reference in the experiments was

delivered by Belcolade (Erembodegem, Belgium). Lipase from Mucor miehei (RM IM) and Novozyme

435 were bought from Novozymes (Bagsvaerd, Denmark).

2. Methods

2.1 Quality of the starting oil

The quality of the starting oil was evaluated by the following methods:

2.1.1 Peroxide value (PV)

AOCS Official Method Cd 8b-90 (1996)

2.1.2 p-anisidine value (p-AV)

AOCS Official Method Cd 18-90 (1996)

2.1.3 Totox value

The Totox value, defined as 2 times the PV + p-AV, was calculated to determine the total oxidation

value (Rossell, 1994).

2.1.4 Acid value and FFA

AOCS Official Method Ca 5a-40 (1966)

2.2 Chemical composition of the starting oil

2.2.1 Fatty acid profile

AOCS Official Methods Ce 1-62 (1990) & Ce 2-66 (1989)


2.2.2 Triacylglycerol profile

2.2.2.1 TAG profile

The TAG profile was determined by using the Shimadzu HPLC in combination with an evaporative

light-scattering detector (ELSD) (Alltech-3300, Alltech Associates Inc., Lokeren, Belgium). The N2 gas

flow rate was set at 1.2 L/min, the nebulizing temperature at 45°C and the acquisition gain was 1.

The fat samples were dissolved in a mixture of 70% acetonitrile (ACN) and 30% dichloromethane

(DCM). The reversed phase C18 column (Grace-Aldrich) was used.

The mobile phase was ACN and DCM. The same elution program was used as described by Rombaut

et al. (2009). The flow was maintained at 0.72 mL/min.

2.2.2.2 Positional isomeric TAGs

Using previous method, it was not possible to separate symmetric and asymmetric TAGs. Therefore,

a second method, with a silver ion column was used to determine the TAG composition. The method

described by Macher & Holmqvist (2001) was adjusted. The TAG profile was determined with the

Shimadzu HPLC in combination with ELSD as detector. Following ELSD conditions were used: a gas

flow rate of 1.5 L/min, the nebulizing temperature of 38°C and the acquisition gain was 1.

The mobile phases were heptane and acetone, the flow rate was 1.0 mL/min. Prior to sample

injection, the column was reconditioned during 12 min at 98% heptane and 2% acetone. After

injection of the sample, the concentration of acetone was increased to 3% in 5 min and kept there

for 5 min. This was followed by a further increase to 10% in 10 min, holding it there for 5 min. Finally,

the acetone concentration was increased to 80% over 10 min. For the sample preparation, the fat

was dissolved in heptane, which was diluted to a concentration of 1 mg/mL. The samples were

analyzed in duplicate.


2.3 The enzymatic acidolysis

Acidolysis reactions with lipase were carried out at different conditions. Reactions were performed in

a glass container, placed in a water bath with mechanical agitation at 300 rpm. In general, substrates

(1 mol oil + necessary quantity of FAM) and water were mixed and heated to the reaction

temperature for 20 min. The reaction started when enzyme was added. Reactions were stopped and

the interesterified oil was filtered through Wathman filter paper No 40 with vacuum to remove

enzymes.

At different time intervals, samples were drawn for analysis. Before taking the samples, the stirrer

should be turned off for 1-2 min in order to let the enzyme particles sediment. Samples were taken

from the top of the oil. Sampling was done with a 1 mL pipette and a 150 mesh metal filter. The

samples were stored in the freezer at -18°C.

The enzyme was washed with acetone to remove all fat residues and to reuse it.

The different reaction parameters that were tested, and their range, are given in table 4.

Table 4: An overview of the tested enzymatic acidolysis parameters and their range.

Parameter Tested range

Reaction time 1 to 72h

Reaction temperature 60 to 75°C

Water content 0 to 5% (based on substrate)

Enzyme load 5 to 30% (based on substrate)

Substrate ratio 1/1 to 1/7 (mol oil/ mol FAM)

2.4 Response surface methodology

The software used to optimize the interesterification reaction through RSM was Design-Expert® 8.0.2

from Stat-Ease Corporation, Minneapolis, USA. A central composite design was applied in this work.

The five factors were reaction temperature (°C), reaction time (h), substrate molar ratio

(HOSO/FAM), water content (% based on the substrate) and enzyme load (% based on the substrate).

Two responses were evaluated. The first was the amount of saturated-unsaturated-saturated (%

SUS) TAGs, mainly POP, POSt, StOSt, formed in each sample. The second one was the amount of

saturated-unsaturated-unsaturated (% SUU) TAGs, mainly POO and StOO, formed.

Using the optimized parameters given by the software, the interesterified oil was produced on a

large scale (figure 9). Larger amounts of interesterification product are necessary to perform the SPD

(see further).


Figure 9: Enzymatic acidolysis reaction on a big scale in optimized conditions.

2.5 Short path distillation Figure 10 represents the different parts of the SPD installation (VTA, Deggendorf, Germany). The

product was distilled two times to reduce the amount of FFA to an acceptable amount.

Figure 10: SPD equipment (Oleon, Belgium).


In table 5 the parameters for the different pumps and water baths are given.

Table 5: Distillation parameters in SPD.

Equipment part Condition

Feed 70°C Evaporator 200°C Residue 60°C Distillate 70°C Vacuum 0.003 mbar Wiper speed 850 rpm Pump for feed 20 Hz Pump for residue (for first distillation) 10 Hz Pump for residue (for second distillation) 18 Hz Pump for distillation 15 Hz

2.6 Fractionation

Two different solvent fractionation methods were evaluated.

The first procedure used, was the solvent fractionation described by Chong et al. (1992). This method

was based on the patented procedure from 1991 by UNILEVER PLC (European patent 0 199 580 B1).

The different steps in the fractionation procedure are given below.

1) Glyceride-hexane solution 1:10 (w/v) at 4°C for 24h

2) Filter off the precipitated fat (vacuum) at 4°C

3) Wash the crystals with hexane at 4°C

4) Evaporate the filtrate to dryness (rotavapor)

5) Filtrate-acetone solution 1:10 (w/v) at 4°C for 24h


7) Wash the crystals with acetone at 4°C

8) Evaporate the precipitate to dryness (rotavapor)

9) Blow nitrogen gas through the product at 60°C for 4h

The second procedure used, is a method described by Chang et al. (1990) and Abigor et al. (2003).

The different steps in the fractionation procedure are given below.

1) Glyceride-acetone solution 1:10 (w/v) at 22°C for 24h


3) Filtrate is cooled to 4°C for 4h


5) Wash the crystals with acetone at 4°C

6) Evaporate the precipitate to dryness (rotavapor)

7) Blow nitrogen gas through the product at 60°C for 4h


2.7 Pulsed nuclear magnetic resonance (pNMR)

A Maran ultra pulsed field gradient NMR (Oxford Instruments, Tubney Woods, Abingdon, UK), 10 mm

diameter NMR tubes (Bruker, Karlsruhe, Germany) and a Water bath (Julabo, Seelbach, Germany)

were used.

The fat samples were melted in the oven for 1h at 70°C before the NMR tubes were filled with 3.5 mL

of the sample. Every sample was analyzed in triplicate.

Two different techniques were used, a non-isothermal method and an isothermal method.

2.7.1 Non-isothermal method (non-tempered)

Initially, the fat has to be melted completely to remove all crystal history. A cooling step is necessary

in the next step, so the fat is completely crystallized. Finally, the fat is held at a defined temperature

for some time to equilibrate at that temperature (Timms, 2003). This procedure is shown below.

1) Oven at 70°C for 60 min

2) Water bath at 0°C for 90 min

3) Water bath at 5°C for 60 min

4) Measure the SFC with the pNMR

5) Increase the temperature of the water bath with 5°C

6) Measure the SFC after 60 min

7) Repeat step 5 and 6 until the SFC content becomes 0%

2.7.2 Non-isothermal method (tempered)

In this method (IUPAC 2.150 serial tempered method), the samples were tempered before measuring

the SFC content by keeping them in a water bath at 26°C for 40h. This was done after step 2 in the

previous procedure. After 40h, the samples were cooled to 0°C for 90 min and the earlier described

procedure was continued from step 3.

2.7.3 Isothermal method

In the first step, the NMR tubes were placed in the oven at 70°C for 60 min in order to melt every

possible crystal present in the sample. Then, the samples were placed into the water bath of 20°C.

Measurements were done at different time intervals.

2.8 Differential scanning calorimetry (DSC)

A Q1000 Differential Scanning Calorimeter (-80 to 400°C) (TA Instruments New Castle, USA) with a

refrigerated cooling system (TA Instruments New Castle, USA) was used. An empty pan was used as a

reference. Pans were filled with 5 to 15 mg of the sample. Every sample was analyzed in triplicate.

Two procedures were used, a non-isothermal method and an isothermal method.


2.8.1 Non-isothermal method

The procedure is shown below.

1) Equilibrate at 65.00°C

2) Isothermal for 10 min

3) Ramp 5.00°C/min to -20°C


5) Ramp 5.00°C/min to 65.00°C

2.8.2 Isothermal method

The procedure is shown below.



3) Ramp 10.00°C/min to 20°C

4) Isothermal for 230 min*

*Slow melting samples were held isothermally for 430 min.

2.9 Polarized light microscopy

The polarized light microscope (PLM) Leitz Diaplan (Pleitz, Wetzlar, Germany) was used together with

the temperature-controlled stage Linkam PE94 (Linkam Scientific Instruments, Surrey, UK) and the

Olympus color view camera (Olympus, Aartselaar, Belgium).

The samples were melted in the oven at 70°C. One drop of every sample was placed on a rest plate

with a Pasteur pipette and covered with a cover plate.

The isothermal crystallization procedure is shown below.


2) Isothermal at 65.00°C for 10 min

3) Cool to 20°C at 10.00°C/min

4) Isothermal at 20°C for 90 min

A picture of the crystals was taken at different time intervals. The samples were kept for 6 weeks at

20°C and an image of the crystallizing fat was made every week.

2.10 Statistical analysis

The data were statistically analyzed with S-plus 8_0 software package. To evaluate significant

differences between data, ANOVA was used. A multiple comparison was performed between

different ratios or contents of one of the parameters. The Tukey test was used to detect significant

differences. All significant differences were based on a 95% significance level (p=0.05). All reactions

were conducted in duplicate.


Results and discussion

1. Introduction CBEs are non-lauric vegetable fats which are completely compatible with CB. Therefore, the CBE can

replace the expensive CB in chocolate. The goal of this research was to produce a CBE by enzymatic

acidolysis of the cheap and commercial available HOSO with a FAM mainly consisting of P and St.

The first part of the research deals with the characterization of the starting oil and the evaluation of

its quality. The chemical characterization consisted of analyzing the FA and TAG composition. To

evaluate the oxidative quality of HOSO, the amount of FFA, PV, p-AV and totox value were

determined.

Secondly, parameters of the enzymatic acidolysis reaction such as reaction time, reaction

temperature, water content, enzyme load and substrate ratio were optimized by RSM in order to

obtain the highest yield of POP, POSt and StOSt. Some trials were performed in order to investigate

the possibility of increasing the product yield by reducing the amount of FFA in the final product. This

was done through the use of glycerol and non-specific enzyme at some point in the enzymatic

acidolysis reaction.

The interesterified product was purified by SPD and fractionation was done in the third step to

increase the purity of the CBE.

In the fourth part of the research, the produced CBE was characterized, chemically and physically.

TAG composition was determined by HPLC and FA profile by GC. Melting and crystallization

properties were analyzed by DSC and pNMR. The produced CBE was mixed in different ratios with CB

in order to analyze the compatibility with CB. Polarized light microscopy (PLM) was applied to analyze

the crystal microstructure of these mixtures.


2. Characterization of HOSO

2.1 Chemical characterization Table 6 gives the results of the different parameters tested to evaluate the oxidative quality of HOSO.

Also the FA composition of the oil is given in this table.

Table 6: Results of the quality tests and the FFA composition of HOSO. The results of the quality parameters are the average of three repetitions, the FA composition is the average of two repetitions.

HOSO

FFA (%) 0.03 ± 0.02

PV 0.32 ± 0.56

p-AV 0.64 ± 0.35

Totox 5.70

C16:0 (% P) 3.97 ± 0.08

C18:0 (% St) 2.84 ± 0.05

C18:1c (% O) 84.25 ± 0.09

C18:2 (% L) 7.76 ± 0.02

C20:0 (% A) 0.18 ± 0.02

C20:1 (%) 0.20 ± 0.02

∑others (%) 0.71 ± 0.04

Oil that is used for enzymatic interesterification, should be of good quality. A fat with a peroxide

value (PV) less than 2 is considered to be freshly produced (Gray, 1978; Robards et al., 1988;

Novozymes, 2011). The amount of peroxides formed, is related to the degree of oxidation of the oil.

Peroxides are the unstable primary oxidation products which will eventually transform into

secondary oxidation products such as aldehydes, ketones, epoxides etc. According to table 6, the

average PV of HOSO was below the maximum limit, therefore it can be considered as a good quality

oil if only the primary oxidation products were taken into account.

It is outmost important that the p-AV is held low. This value gives the amount of secondary oxidation

products present in the oil, indicative of the bad quality of the oil. For this reason, the p-AV may not

be neglected when making a conclusion about the oxidation status of the fat. According to the

protocol provided by Novozymes, the p-AV should be lower than 4 in order to prevent reactions

between the amino acids in the lipase-backbone and the secondary oxidation products. These

reactions can lead to loss of activity of the enzyme (Novozymes, 2011). HOSO met this specification

(table 6).


The totox value indicates the total oxidation of the fat sample. It uses both the PV and the p-AV

(O’Keefe & Dike, 2010). The acceptable level of the Totox value should be below 10 (Podmore, 1990)

which was the case for the starting oil.

HOSO contained an average amount of FFA of 0.03%. This low amount of FFA indicates a high quality

of the substrate.

The FA composition is given in table 6. From the table it is clear that HOSO contained a high amount

of oleic acid (C18:1). In the synthesis of a CBE, a suitable starting oil should have a high level of oleic

acid (Khumalo et al., 2002). This means that HOSO is a good substrate for CBE production.

The TAG composition is presented in table 7. HOSO consisted mainly of OOO (62.09%) and

considerable amounts of POL (7.66%), POO (11.01%) and StOO (8.43%). There were also small

amounts of LOL, LOO and StOL present.

Table 7: TAG composition of HOSO with P: palmitic acid, St: stearic acid, O: oleic acid and L: linoleic acid.

TAG HOSO

LOL 2.47 ±0.09

LOO 2.99 ±0.09

POL 7.66 ±0.10

StOL 1.35 ±0.02

OOO 62.09 ±0.87

POO 11.01 ±0.14

StOO 8.43 ±0.14

POSt ND

PPSt ND

StOSt ND

PStSt ND

*ND: not detected


2.2 Physical properties of HOSO The crystallization and melting behavior of the starting oil are measured by DSC and pNMR.

2.2.1 Non-isothermal crystallization and melting behavior as measured by DSC

The non-isothermal method was executed as was described in paragraph 2.8.1 in ‘Materials and

methods’.

In figure 11, the crystallization and melting profile of HOSO, obtained with DSC non-isothermal

method, is shown. According to figure 11, the melting curve shows a broad peak at low temperatures

(-5°C), this means that the oil was completely liquid at room temperature.

Figure 11: Non-isothermal DSC results of HOSO.

2.2.2 Solid fat content as measured by pNMR

HOSO was liquid (0% SFC) at 5°C, this is due to the high amount of triolein (OOO), which causes HOSO

to be a low melting oil.


3. Enzymatic acidolysis

3.1 Optimization of the reaction conditions The effect of each parameter (reaction time, reaction temperature, water content, enzyme load and

substrate ratio) in the acidolysis reaction on the amount of desired TAGs, was analyzed separately

while the other parameters were held constant. An overview of the experimental design is illustrated

in figure 12. The selection of the best conditions was based on the percentage of POP + POSt + StOSt,

the three main TAGs in CB.

Figure 12: Process scheme to find the optimum conditions for the enzymatic acidolysis reaction.

•Time: 1, 2, 3, 4, 6, 8, 24, 30, 48, 56 and 72h

•Temperature: 70°C

•Water content: 1% (based on substrate)

•Enzyme load: 10%

•Substrate ratio : 1/7: HOSO / FAM (mol / mol)

Effect of reaction time

•Time: Best that was found in the first step

•Temperature: 60, 65, 70, 75°C

•Water content: 1%

•Enzyme load: 10%


Effect of reaction temperature


•Temperature: Best that was found in the second step

•Water content: 0%, 1%, 3%, 5% (based on substrate)

•Enzyme load: 10%


Effect of water content



•Water content: Best that was found in the third step

•Enzyme load: 5%, 10%, 15%, 20%, 25%, 30%


Effect of enzyme load



•Water content: Best that was found in the third step

•Enzyme load: Best that was found in the fourth step

•Substrate ratio : HOSO / FAM (mol / mol): 1/1, 1/2, 1/3, 1/4 , 1/5, 1/6, 1/7, 1/8, 1/9, 1/10

Effect of substrate ratio


3.1.1 Reaction time

The first parameter tested, was the reaction time. The substrate ratio was set at 1:7 (mol HOSO: mol

FAM), the water content was 1% (based on the substrate), 10% enzyme load was used (based on the

substrate) and the temperature was held at 65°C. Samples were drawn after 1, 2, 3, 4, 6, 8, 24, 30,

48, 56 and 72h. The average percentage of the main TAGs after two repetitions is given in figure 13.

The amount of TAGs increased until 8h of reaction. The reaction between OOO and FFA is a

reversible reaction. From the beginning until 4h, this reaction shifted more to the product side;

meaning that the rate of acidolysis was higher than hydrolysis. From 4 to 8h, the reaction reached its

equilibrium and the rate of both acidolysis and hydrolysis became the same. But when the reaction

continued after 8h, the hydrolysis reaction became more dominant than the acidolysis. This means

that the optimum reaction time was between 4 to 8h.

From figure 13, it was obvious that a choice should be made between 4, 6 and 8h. The amount of

trisaturated TAGs (SSS), which is the sum of StStSt, PPSt and PStSt, increased from 8h onwards.

Therefore, this reaction time was not the optimum one. The increase in SSS TAGs after longer

reaction times was also noticed by Palla et al. (2012). These SSS TAGs are undesirable because they

will increase the melting point of the end product (Chong et al., 1992; Chang et al., 1990). They are

unwanted when CBE is added to chocolate as it should melt around body temperature.

Statistics was used to determine the optimum sampling time. There is no significant difference

(p>0.05) between 4 and 6h. Khumalo et al. (2002) concluded that after 4h, the FA were incorporated

in desired amounts while Ciftci et al. (2009) found an equilibrium in TAG formation after 6h. Based on

these findings, a reaction time of 6h was selected for further analysis.


Figure 13: The % TAG (POP, POSt and StOSt) and trisaturated TAGs (SSS) at different sampling times.

3.1.2 Reaction temperature

The effect of temperature is illustrated in figure 14. Four different temperatures were tested. The

percentages of the desired TAGs at different temperatures are given. The amount of the three main

TAGs (POP, POSt and StOSt) decreased when the temperature was increased to 75°C. From this

graph, it can be concluded that a temperature of 70°C gave significantly higher amounts of desired

TAGs. 70°C is also the optimum temperature of the enzyme that was used (Novozymes, 2011).

All investigated temperatures were significantly different (p<0.05). Consequently, for further CBE

preparation, a temperature of 70°C was chosen.

Ciftci et al. (2008), also reported that lower temperatures and shorter reaction times are

economically advantageous and it prevents acyl migration. In acyl migration, FA can migrate from the

sn-1,3 position to the sn-2 position or vice versa. According to Palla et al. (2012), a higher reaction

temperature results in a higher amount of SSS TAGs formed by acyl migration. The acyl migration is a

problem because it changes the crystallization behavior and melting profile of the end product

usually in an undesirable way.

0

10

20

30

40

50

60

70

80

90

1 2 3 4 6 8 24 30 48 56 72

TA

G (

%)

Sample time (h)

SSS

StOSt

POSt

POP


Figure 14: The % TAG (POP, POSt and StOSt) using different temperatures. The results are the average of two

repetitions.

3.1.3 Water content

The effect of different percentages of water was studied. The three main TAGs (POP, POSt and StOSt)

and the amount of by-products (MAG + DAG) are given in figure 15. As can be seen from the graph,

the highest amount of desired TAGs was formed when 3% water was added. But regarding the

amount of MAG and DAG, which should be as low as possible, adding 1% water gave better results.

Enzymes need a certain amount of water to be active and to be able to hydrolyze the fatty acid from

the glycerol backbone on the sn-1 and sn-3 position.

The obtained p-value (=0.30) indicated that there was no significant difference in amount of POP,

POSt and StOSt formed between the used water contents. The amounts of MAG and DAG formed

between 1% and 3% water content were significantly different. A water addition of 1% was chosen as

this gave the lowest amount of these by-products.

Rodrigues & Fernandez-Lafuente (2010) concluded the same in their report. For the activation of

lipases, a certain amount of water has to be added, but adding too much water will shift the reaction

to hydrolysis instead of synthesis. To find the optimum water content; the type of lipase, support and

substrate are important. Sellappan & Akoh (2000) and Tchobo et al. (2009) stated that the used

enzyme, RM IM, was applicable in systems where the water activity was held low. In a system with a

low water activity, the enzyme performed active and stable.

0

10

20

30

40

50

60

70

60 65 70 75

TA

G (

%)

Temperature (°C)

StOSt

POSt

POP


Figure 15: The % TAG (POP, POSt and StOSt) and the amount of MAG + DAG formed, using different water

contents. The results are the average of 2 repetitions.

3.1.4 Enzyme load

The next parameter that was investigated, was the effect of the amount of enzyme on the

percentage of desired TAGs. Enzyme concentrations varying from 5 to 30% were tested. In the report

of Ciftci et al. (2008), it was found that the amounts of POP and StOSt that were formed were almost

equal, therefore, the same amounts of P and St were incorporated. Consequently, the lipozyme IM

had no selectivity for P or St acid.

In general, when more enzyme was added, more of the desired TAGs were formed (figure 16).

However, adding more than 10% of the enzyme had a limited effect on the amount of desired TAGs.

The reduction of the % TAGs at higher amounts of enzyme was probably due to the increase of solid

particles in the reaction. This will result in an increasing viscosity of the mixture in a way that the

interaction of the reactants with each other decreases, causing a low incorporation of FFA to the

glycerol backbone and reducing the amount of desired TAGs. The increased amount of enzyme

limited the stirring rate, resulting in a bad mixing of the reactants.

Wang et al. (2006) came to the same conclusion, they found that an increased enzyme load

accelerated the reaction rate and improved the incorporation of the fatty acids, but this relationship

was not linear. From figure 16, an enzyme load of 10, 15 or 20% should be chosen as they gave the

highest amounts of TAGs. Since there was no significant difference between these different amounts

of enzyme, 10% was chosen from an economical point of view.

0

10

20

30

40

50

60

70

0 1 3 5

TAG

(%

)

Water content (%)

MAG+DAG

StOSt

POSt

POP


Figure 16: The % TAG (POP, POSt and StOSt) using different enzyme loads. The results are the average of two

repetitions.

3.1.5 Substrate ratio

Different substrate ratios of HOSO and FAM were tested. These substrate ratios ranged from 1:1 to

1:10 (mol HOSO: mol FAM). From figure 17 it is clear that when the amount of FFA in the mixture

increased, the TAG formation also increased. This was in accordance with the results of Xu (2003).

Other studies of Wang (2006) and Hoy & Xu (2001), concluded that the percentage of conversion

increased when the substrate ratio raised up to a point where further increase of the substrate ratio

resulted in a decrease of the amount of TAGs. A high amount of FFA will acidify the enzyme layer

because of the high level of free or ionized carboxylic acid groups which causes the enzyme to be

inactivated (Ciftci et al., 2008).

All substrate ratios were significantly different except for ratios 1:8 and 1:9. From figure 17 it is clear

that substrate ratios 1:7 through 1:10 gave the highest amount of desired TAGs. Due to cost

considerations and ease of working, substrate ratio 1:7 was selected for CBE preparation.

0

10

20

30

40

50

60

70

5 10 15 20 25 30

TA

G (

%)

Enzyme (%)

StOSt

POSt

POP


Figure 17: The percentage of the TAG (POP, POSt and StOSt) for different substrate ratios. The results are the

average of two repetitions.

3.1.6 Improving the yield of the reaction

Enzymatic acidolysis of HOSO with FAM in optimized reaction conditions resulted in a low yield of the

desired TAGs (POP, POSt and StOSt), since 64.78% of FFA remained. Several methods were

considered to increase the yield of desired TAGs. In the next part, the effect of glycerol addition in

order to reduce the amount of FFA was investigated. In the last trial, a non-specific enzyme was

added to reduce the amount of FFA and undesirable DAGs and MAGs.

Addition of glycerol

To shift the reaction to the product side, glycerol was added after 4h in different molar ratios of 1:1

to 1:4 to the acidolysis reaction. Figure 18 (A) displays the decreasing amount of FFA from 64.78% to

8.68% by adding 4 mol glycerol. On the other hand, there was an obvious increase in the amount of

DAGs formed. This is due to the sn-1,3 specific enzyme which can only affect the sn-1,3 position of

the glycerol backbone. The sn-2 position remained unchanged, leading to the creation of 1,3-DAGs.

Another cause of DAG formation is acyl migration. Figure 18 (B) gives the percentage of the three

main TAGs (POP, POSt and StOSt) at different ratios of glycerol. The amount of POP, POSt and StOSt

increased when glycerol was added in a ratio of 1:1 and 1:2 (mol HOSO: mol glycerol). However, at

higher ratios of glycerol, this amount decreased. The addition of more glycerol, probably reduced the

amount of FFA to the level that the enzyme started to attack the present TAGs and hydrolyzed the

fatty acids on the first and third position on the glycerol backbone.

0

10

20

30

40

50

60

70

80

1:1 1:2 1:3 1:4 1:5 1:6 1:7 1:8 1:9 1:10

TA

G (

%)

Substrate ratio (mol/mol)

StOSt

POSt

POP


According to Fadiloglu et al. (2003), the FFA content of an oil can be reduced by enzymatic

glycerolysis. In their research, an immobilized lipase from Candida antarctica, Novozyme 435, was

used. It was also noticed that an accumulation of water favored the reverse reaction, the hydrolysis

of the TAGs. This may explain the decrease in amount of TAGs when more glycerol was added.

During the esterification reaction of FFA and glycerol, water is released and an excess of water will

result in more hydrolysis of the formed glycerides.

Based on the observed results, the best option was to add only 2 mol of glycerol in order to reduce

the amount of FFA to 35.65% without the formation of large amounts of DAGs.

Figure 18: The percentage of FFA, DAG and TAGs (A) and a detail of the desired TAGs (POP, POSt and StOSt)

(B) for different ratios of glycerol added to HOSO.

Combined addition of glycerol and non-specific enzyme

When only glycerol was added to the acidolysis reaction, a large amount of DAGs was formed. To

transform these DAGs to TAGs, trials were performed in which non-specific enzyme was used. Three

different methods were tested to add glycerol and non-specific enzyme, Novozyme 435. The

different trials are schematically presented in table 8. The total reaction time of each trial was 8h.

0

10

20

30

40

50

60

70

80

0 1 2 3 4

%

Mol glycerol added per mol HOSO

FFA

DAG

TAG

0

5

10

15

20

25

30

0 1 2 3 4

TAG

(%

)


StOSt

POSt

POP

A B


Table 8: Three different methods in which glycerol and Novozyme 435 were added to the acidolysis reaction.

Reaction time (h) Method A Method B Method C

0 HOSO: FAM (1:7) HOSO: FAM (1:7) HOSO: FAM (1:7)

4 +Glycerol 1:1 to 1:4*

+8% Novozyme 435**

+Glycerol 1:1 to 1:4*

/

6 / +8% Novozyme 435**

-Remove RM IM

+Glycerol 1:1 to 1:4*

+8% Novozyme 435**

8 End reaction End reaction End reaction

* mol HOSO: mol glycerol

** w/w glycerol + FFA

The obtained amounts of FFA, DAGs and TAGs (POP, POSt and StOSt) are given in figures 19 and 20.

In general, the amount of FFA (figure 19 (A)) with the three methods decreased when more glycerol

and the non-specific enzyme was added. However, the amount of remaining FFA after the three

methods using the non-specific enzyme, was still larger than when only glycerol was added to the

interesterification reaction. This was probably due to the interference of both enzymes with each

other. When analyzing the amount of remaining FFA for a particular method at the different ratios of

glycerol added, no significant difference (p>0.05) was observed. This was the case for all three

methods with Novozyme 435 and glycerol. This was in contrast to the significant decrease in FFA

when only glycerol was added in different ratios.

In conclusion, method A resulted in a lower amount of FFA and DAGs formed compared to method B.

Method C was preferred as it gave the lowest amount of FFA remaining when comparing the three

methods (except for molar ratio 2). When applying method C, a maximum of 1 mol of glycerol should

be added as this gave a reduction of FFA from 64.78% to 44.53% and a production of 38.62% of DAG.

Generally, the amount of DAGs for all three methods increased when more glycerol and non-specific

enzyme was added (figure 19 (B)). Adding 3 or 4 mol of glycerol showed no significant difference

(p>0.05) in amount of DAGs produced. When only glycerol (1 or 2 mol) was added to the reaction, a

lower amount of DAGs was obtained compared to the trials using the combination of glycerol and

non-specific enzyme.


Figure 19: The percentage of FFA (A) and DAG (B) for different ratios of glycerol and non-specific enzyme

added to HOSO.

The total amount of TAGs (POP, POSt and StOSt) obtained (figure 20), was significantly different

depending on the amount of glycerol and Novozyme 435 added. It can be concluded that the amount

of desired TAGs for all three trials decreased when more glycerol and non-specific enzyme was added.

The largest decrease in TAGs was obtained with method B. The amount of TAGs in these sets of

experiments decreased more than when only glycerol was added to the reaction.

In conclusion, adding glycerol resulted in the formation of DAGs and a reduction of FFA. On the other

hand, in the presence of excess glycerol, the enzyme started to hydrolyze the fatty acids of the

desired TAGs leading to a decrease of POP, POSt and StOSt. Taking into account that the removal of

DAGs is more difficult than FFA, using glycerol in combination with non-specific enzyme is not a good

strategy to improve the yield of the reaction.

0

10

20

30

40

50

60

70

0 1 2 3 4

FFA

(%

)


Method A

Method B

Method C

0

10

20

30

40

50

60

70

0 1 2 3 4

DA

G (

%)


Method A

Method B

Method C

A B


Figure 20: The percentage TAG (POP, POSt and StOSt) for different ratios of glycerol and non-specific enzyme.

Method A (A), method B (B) and method C (C).

3.2 Optimization of the reaction parameters by RSM

Previous determination of the parameters in the acidolysis reaction did not take into account the

possible interaction between several parameters. Therefore, RSM is used to determine the optimized

reaction time, reaction temperature, water content, enzyme load and substrate ratio when also the

interactions are considered. The goal is to maximize the amount of SUS TAGs and minimize the

amount of SUU TAGs formed during the reaction.

3.2.1 Experimental design

Based on the obtained results by lipase-catalyzed acidolysis of the HOSO on small scale, RSM was

applied to optimize further the value of each reaction parameter. These parameters are substrate

ratio (A), enzyme load (B), water content (C), temperature (D), and reaction time (E). Table 9 gives

the ranges of each parameter acquired after small scale reaction.

0

5

10

15

20

25

0 1 2 3 4

TA

G (

%)


StOSt

POSt

POP

0

5

10

15

20

25

0 1 2 3 4

TA

G (

%)


StOSt

POSt

POP

0

5

10

15

20

25

0 1 2 3 4

TAG

(%

)


StOSt

POSt

POP

A B

C


Table 9: The five factors used for RSM with the unit and lower and upper limit.

Name Unit Low Level (-1) High Level (+1)

A: substrate ratio mol 6 8

B: enzyme load % 8 12

C: water content % 1 3

D: temperature °C 65 70

E: reaction time h 4 8

There were 47 runs, including 5 center points, which were performed in random order. Two

responses were evaluated, the percentage of the three most important SUS TAGs that were formed

(POP, POSt and StOSt), and the sum of SUU TAGs (mostly POO and StOO) produced during the

interesterification reaction. The aim was to maximize the % SUS and to minimize the % SUU.

Table 19 in appendix gives the experimental amount of SUS and SUU for the runs with the different

levels of the parameters. These quantities are based on the total amount of SUS and SUU analyzed by

HPLC.

3.2.2 Model fitting

The obtained experimental results of SUU and SUS were inserted into the central composite design

for further analysis. The best model was selected based on three statistical criteria: the sum of

squares, Lack-of-fit, and model R². Based on these results, a quadratic model was selected. First, the

responses were fit to the factors by multiple regression. A regression analysis, with backward

elimination, was performed in order to explain the responses as function of the factors, and to

remove interaction between factors which did not contribute significantly to the model. The

backward regression procedure makes every term to fit the data, followed by considering the

remaining candidate factors. The backward method is considered to be the most robust choice for

algorithmic model reduction because all model terms will be given a chance to be included in the

model (De Clercq, 2011).

Table 19 in appendix not only shows the actual experimental results of % SUS and % SUU, but also

the predicted amounts. In table 10 the regression coefficients of the different factors are shown with

the corresponding p-value. When a p-value of 0.1 was chosen, all parameters showed a significant

effect, except for the influence of water on the amount of SUU. Although the factor for the water

content had an insignificant effect on the SUU response (p>0.1), it was not removed by backward

elimination to maintain the hierarchy of the model. When a p-value of 0.05 was chosen; A, B, D, E,

B*D, C*D and D² were significant model terms. This means that the interactions water content-

temperature and enzyme load-temperature are significant.


Table 10: Regression coefficients of the quadratic model for the response variables.

Variables Coefficient Degree of freedom p-value

SUS SUU

SUS SUU

constant 58.38 35.82 1 - -

A 3.13 -2.41 1 <0.0001a 0.0002a

B 1.91 -1.39 1 0.0101a 0.0237a

C 1.24 -0.96 1 0.0886b 0.1135NS

D -5.26 3.95 1 <0.0001a <0.0001a

E 2.43 -2.14 1 0.0015a 0.0008a

B*D 3.49 -2.88 1 0.0001a 0.0002a

C*D 3.15 -2.49 1 0.0005a 0.0009a

B² -1.20 0.93 1 0.0641b 0.0853b

D² -1.43 1.52 1 0.0287a 0.0061a

NS: not significant at p>0.1

a: significant at p<0.05

b: Significant at p<0.1

Table 11 gives the ANOVA analysis of the experiment. It states that the quadratic terms are

significant at p<0.05, and the Lack-of-fit or the adequacy test is not significant at p<0.1. The quadratic

model showed a R² of 0.79 and 0.77 for SUS and SUU respectively, which are considered acceptable.

As a result, the model can be used to navigate the design space.

Table 11: Analysis of variance (ANOVA) for the response surface quadratic model.

Source

Degree of

freedom Sum of squares Mean squares F-value p-value

SUS SUU SUS SUU SUS SUU SUS SUU

Quadratic 9 2978.24 1871.49 330.92 207.94 15.31 13.77 <0.0001a <0.0001a

Residual 37 799.84 558.83 21.62 15.10 - - - -

Lack of Fit 33 643.30 431.92 19.22 13.09 0.46 0.41 0.9038NS 0.9323NS

Pure Error 4 165.54 126.91 41.38 31.73 - - - -

Total 46 3778.07 2430.32 - - - - - -

NS: not significant at p>0.1

a: significant at p<0.05

b: Significant at p<0.1


3.2.3 Main effects and interactions between parameters

Table 10 summarizes the multiple regression coefficients, obtained by the use of a least squares

technique to predict a second-order polynomial model for the CBE production. It shows that the SUS

response was positively affected by all parameters except by the temperature. In the case of SUU, all

parameters, when being increased, had a negative effect with the exception of the water content

which affects this response positively. Among all, the temperature had the highest effect followed by

substrate ratio, reaction time, enzyme load, and water content. However, all differences in effect

between parameters are rather limited.

In figure 21, the perturbation plots, or main effect plots, for SUS and SUU are shown. A perturbation

plot assists to compare the effect of all the factors at a particular point in the design space. The

responses were plotted by changing only one factor over its range, while all the other factors were

held constant. The reference point was set at the midpoint (coded 0) for every factor. It was noticed

that the effect of the substrate ratio (A) and reaction time (E) were very similar. Both of them had a

positive linear relation with the SUS response, and a negative linear relation with the SUU response.

Comparing both plots, it was possible to see the opposite effect of each parameter towards SUS or

SUU.

Figure 21: Perturbation plot of SUS (left) and SUU (right) with A: substrate ratio (7 mol); B: enzyme load

(10%); C: water content (2%); D: temperature (65°C) and E: reaction time (6h).

The next figures present the contour plots of the significant interaction terms on the two responses.

In each part of the figure the effect of two parameters on the % SUS or % SUU is shown. At the same

time, the other factors were maintained at their center values. The interaction terms are water-

temperature and temperature-enzyme. Figure 22 shows the interaction between temperature and

enzyme. Figure 23 gives the interaction between temperature and water for SUS and SUU,

respectively. It was found that both interaction terms had a positive effect on SUS but a negative

effect on SUU.


Figure 22: Contour plot of the interaction between enzyme and temperature on % SUS (left) and % SUU

(right).

Figure 23: Contour plot of the interaction between water and temperature on % SUS (left) and % SUU (right).

3.2.4 Optimization

The Design Expert software was used to generate the optimum reaction parameters, aiming to

maximize the yield of SUS and minimize the amount of SUU formed. Table 12 shows the four

optimized reaction conditions generated by Design Expert. The optimization was performed

numerical with response SUS being maximized and SUU being minimized. The parameters substrate

ratio and enzyme load were set at the middle level due to cost consideration, and water was set at

the lowest level. Reaction time and temperature were set within the range of 4-8h and 65-70°C,

respectively.

The following optimal conditions (table 12) were chosen for maximal amount of SUS (70%) with

minimal amount of SUU (26%): Substrate (7-7.99:1 molar ratio), enzyme load (8.20–8.54%), water

content (1%), temperature (65°C), and reaction time (7.97-8h). The corresponding contour plots for

SUS and SUU, when using optimum condition number one, are shown in figure 24. For further

research, optimization number one was retained.


Table 12: Optimum conditions for each combination of parameters and predicted amounts of SUS and SUU given by RSM.

Number Substrate

Ratio

Enzyme

Load

Water

content

Temperature Time % SUS % SUU

1 7.99 8.54 1.00 65 7.98 70.14 26.76

2 7.99 8.50 1.00 65 7.97 70.12 26.77

3 7.98 8.24 1.00 65 7.99 70.08 26.77

4 7.00 8.20 1.00 65.01 8.00 70.07 26.76

Figure 24: Contour plot for the optimal factor levels of % SUS (left) and % SUU (right).

3.2.5 Model verification

For the lipase-catalyzed acidolysis reaction on large scale, the optimized results for the five

parameters were chosen as stated earlier. Several batches were performed and the obtained

interesterified product was analyzed for % SUS and % SUU by HPLC. Table 13 compares the observed

amount of SUS and SUU with the predicted quantity generated by Design Expert for four repetitions.

In conclusion, both observed results for SUS and SUU were very close to the predicted amount after

optimization by RSM.

Table 13: Model verification.

Production no. SUS SUU

observed predicted observed predicted

1 70.0 70.1 24.9 26.7

2 68.3 70.1 25.9 26.7

3 67.2 70.1 26.4 26.7

4 65.5 70.1 23.1 26.7


4. Product purification

4.1 SPD The end product after interesterification, using the optimized conditions, still contained 64.8% FFA.

Therefore, in order to be able to use it as CBE, these FFA should be removed from the product. SPD

was used for this purpose. It was performed in two steps to increase the quality of the product.

During the first step, the amount of FFA was reduced to 12.72%. The amount of FFA in a food

product, should be maximum 0.3% (Paquot & Hautfenne, 1984). Therefore, a second distillation step

was done to remove as much FFA as possible. Titration of the final product showed 0.32% of FFA. The

TAG composition of the product after SPD is presented in table 14. After SPD, no DAGs and MAGs

could be detected in the purified product.

The total sum of POP, POSt and StOSt obtained after SPD was 59.81%. This amount is lower than

predicted by RSM. Probably, during SPD, some of the POP, POSt and StOSt are lost with the removal

of FFA as was also noticed in the report of Lin & Yoo (2009).

Table 14: TAG composition of the product after SPD.

TAG %

StOL 0.20 ±0.01

POL 0.68 ±0.08

OOO 2.23 ±0.21

POO 12.70 ±0.46

POP 12.31 ±0.10

StOO 14.99 ±0.39

POSt 31.91 ±0.34

PPSt 2.65 ±0.08

StOSt 15.59 ±0.81

PStSt 2.86 ±0.26

StStSt 0.67 ±0.09

4.2 Fractionation Fractionation is an important step in the production of a CBE. The CBE should have the same

crystallization, texture and melting properties as real CB. That is why inadequate removal of SSS TAGs

during fractionation will lead to a waxiness feeling when the chocolate melts in the mouth. When

SUU and tri-unsaturated (UUU) TAGs are not removed properly, the SFC profile will be affected.

Finally, the crystallization performance of the CBE will be influenced when the DAGs are not properly

removed (Kellens et al., 2007). Two solvent fractionation methods were evaluated: method A and

method B. A scheme of method A, with the use of hexane and acetone, and method B, in which only

acetone is used as a solvent at two different temperatures, is given in figure 25.


Figure 25: Scheme of the obtained fractions after fractionation using method A or B.

To evaluate both methods, the TAG composition in each fraction was analyzed. The starting product

is defined as the product that was obtained after SPD. The fractions of method A consisted of

following TAGs. The first stearin fraction (SF1) consisted of 24.23% of PPP, PPSt, PStSt and StStSt. The

second olein fraction (OF2) contained 72.26% of OOO, POO and StOO and the second stearin (SF2)

fraction, which should be the CBE, consisted of 83.98% of POP, POSt and StOSt but there was still

9.13% of OOO, POO and StOO remaining. This amount is high compared to CB which has 1.64% of

POO and StOO and 89.5% of POP, POSt and StOSt. The yield of the fractionation process was 48.02%.

This makes the total yield of the process after SPD and fractionation 15.37%. The yield is too low to

consider using this process on an industrial scale.

After using method B, the first stearin fraction (SF1) consisted of 29.69% of PPP, PPSt, PStSt and

StStSt. The second olein fraction (OF2) contained 75.23% of OOO, POO and StOO and the second

stearin (SF2) fraction, which should be the CBE, consisted of 67.28% of POP, POSt and StOSt but there

was still 27.22% of OOO, POO and StOO remaining.


The results of the two fractionation methods were evaluated by comparing the TAG composition of

their three fractions. Figure 26 shows the results for SF1, OF2 and SF2. Comparing two methods, SF1

(figure 26 (A)) in method A contained significantly less (p<0.05) SSS TAGs. The amount of UUU and

SUU TAGs found in OF2 (figure 26 (B)) did not significantly (p>0.05) differ between method A and B.

The percentage of POP, POSt and StOSt in SF2 (figure 26 (C)) of method A was significantly (p<0.05)

higher compared to method B. The amount of UUU and SUU TAGs remaining in SF2 (figure 26 (C))

was significantly lower (p<0.05) for method A compared to method B. This is important for the

melting profile of the produced CBE, since too much UUU and SUU TAGs will result in a soft CBE

which is undesirable.

According to these results, method A was preferred to method B because it gave higher amounts of

POP, POSt and StOSt and the remaining low melting TAGs were lower in the final product, which is

more suitable to use in chocolate and confectionery products.

Figure 26: Percentage of SSS TAGs in SF1 (A), UUU and SUU TAGs in OF2 (B) and SUS, UUU and SUU TAGs in

SF2 (C) compared between two fractionation methods.

0

2

4

6

8

10

12

14

A B

TAG

(%

)

Fractionation method

PPP

PPSt

PStSt

StStSt

0

5

10

15

20

25

30

35

40

45

A B

TAG

(%

)


OOO

POO

StOO

0

10

20

30

40

50

60

A B

TAG

(%

)


POP

POSt

StOSt

OOO

POO

StOO

A

A

B

C


4.3 Physical characterization To evaluate the melting behavior of the different fat samples, DSC was used. The SFC is determined

with pNMR. The melting behavior of the interesterified product, purified product after SPD and

different fractionations of products was analyzed.

The difference in physical characteristics between CB and CBE, which is the SF2 fraction after

fractionation method A, is discussed.


The crystallization and melting profiles, obtained with DSC non-isothermal method are shown in

figure 27 (the product and the purified product after SPD) and in figure 28 (results after

fractionation).

The upper curve in these figures represents the crystallization of the sample during cooling and the

lower part represents the melting curve during heating of the sample. Only the lower part, the

melting curve, will be considered and thereof the following parameters were deduced:

Tonset (°C): the intersection of the baseline with the highest tangent of the curve

Tpeak (°C): the temperature at which the melting curve reaches its peak maximum

Melting heat (J/g): the heat taken up by the fat while melting, this is calculated by

integrating the area of the total melting peak

Width at half height (°C): the width of the peak at half height of the melting peak

The results of the parameters after integration of the melting peaks, are given in table 15.


Table 15: Parameters Tonset (°C), Tpeak (°C), meting heat (J/g) and width at half height (°C) of DSC melting profile (non-isothermal).

Sample Peak

(figure) Tonset (°C)

Tpeak (°C)

Melting heat (J/g)

Width at half height (°C)

Interesterified product

A -3.82 ± 0.01 1.94 ± 0.16 31.25 ± 0.91 4.71 ± 0.17 B 39.00 ± 0.12 50.15 ± 0.11 77.27 ± 2.60 8.66 ± 0.12

Purified product (after

SPD)

A2 -0.66 ± 0.42 12.28 ± 0.28 37.18 ± 2.68 8.89 ± 0.28 B2 26.65 ± 0.55 35.53 ± 0.50 9.50 ± 0.83 11.53 ± 0.52

SF1 A1 -1.74 ± 0.12 11.81 ± 0.50 23.88 11.11 B1 30.72 ± 0.12 39.55 ± 0.26 25.68 ± 0.15 12.64 ± 0.20

OF2 -10.9 ± 0.16 7.91 ± 0.42 33.77 ± 1.20 15.26 ± 0.32

SF2 A2 12.58 ± 0.23 18.13 ± 0.06 33.79 ± 1.85 5.67 ± 0.18 B2 30.29 ± 0.44 37.64 ± 0.30

9.16 ± 0.45 7.85 ± 0.06

CB

14.05 ± 0.04 21.45 ± 0.15 77.76 ± 5.37 6.59 ± 0.08

OF2*

-10.7 ± 0.12 6.05 ± 0.00 22.5 ± 1.09 13.53 ± 0.09

SF2*

A2* 1.73 ± 0.66 12.23 ± 0.18 -0.37 ± 0.03 7.42 ± 0.30

B2* 16.74 ± 0.18

C2* 27.51 ± 0.12 34.67 ± 0.13 8.59 ± 0.66 11.66 ± 0.55

*: fractionation method B.

The product (figure 27) gave two peaks of which A is the peak of the TAGs and peak B represents the

melting point of the FFA present in the interesterified product. The total amount of FFA was

completely melted at 60°C. The low onset temperature (-3.82°C) of peak A indicated that there were

also low melting SUU and UUU TAGs present in the product. The peak of FFA in the purified product

was almost completely eliminated and shifted towards lower temperatures (35.53°C) indicating the

presence of SSS TAGs (B2). A2 stands for the mix of SUS TAGs (CBE) and the SUU TAGs present in the

product after SPD.


Figure 27: Non-isothermal crystallization and melting profile of the interesterified product (product) and the

purified product (after SPD) as measured by DSC.

Figure 28 shows the crystallization and melting graphs of each fraction obtained by fractionation

method A. In figure 29, a comparison is made between the final fraction (SF2) of the two

fractionation methods.

SF1 (method A), contained SSS TAGs resulting in a peak B1 at maximum 39.55°C. Peak A1 indicates

that a small amount of other, low melting (11.81°C), TAGs were lost during this fractionation

procedure. The OF2 (method A) contained the SUU and UUU TAGs (POO, StOO and OOO) resulting in

a peak at 7.91°C. The final fraction SF2 (method A) had a large peak around 20°C which indicated the

presence of the desired TAGs (POP, POSt and StOSt). Peak B2 (37.64°C) showed that there was still a

small amount of SSS TAGs present in the SF2 fraction.

Figure 28: Non-isothermal crystallization and melting profile of the fractions after fractionation method A as

measured by DSC.

A

B

A2

B2

A2

B2

A1 B1


When comparing the SF2 fractions of the two fractionation methods (figure 29), one can notice the

clear difference in the A2 (method A) and A2* (method B) peaks. The latter method resulted in a

higher amount of SUU TAGs (peak A2*) present in the sample, whereas mehod A resulted in a SF2

fraction closer resembling CB. This was also concluded by comparing the peak temperatures: 18.13°C

(A2) and 12.23°C (A2*), the lower temperature for peak A2* indicated that there were more SUU

TAGs left after method B. B2* (method B) showed the lower amount of POP, POSt and StOSt present

in SF2* (method B) compared to SF2 (peak A2). Consequently, based on the results obtained from

the DSC graphs, fractionation method A seems the most effective.

Figure 29: Non-isothermal crystallization and melting profile of the SF2 fractions after fractionation method A and B as measured by DSC.

4.3.2 SFC

Comparisons are made between the SFC of the interesterified product, the purified product after

SPD, different fractions of products and CB. Figure 30 shows the results of the pNMR non-isothermal

method for the following tempered and non-tempered samples: the interesterified product (product),

the product after SPD (purified product), the second stearin fraction (SF2) and CB.

The product has high contents of FFA and SSS TAGs, that is why this sample needs very high

temperature to melt completely (60°C). These results were also confirmed by DSC that showed the

complete melting of the FFA present in the product at 60°C. After SPD, the FFA were removed and

the melting point of the obtained product reduced (45°C), but this melting point remained higher

compared to CB due to the presence of SSS TAGs.

A2* B2*

C2*

A2

B2


The product after fractionation, using method A (SF2), showed a similar melting profile as CB but it

had lower SFC till 30°C. Between 30 to 40°C, the amount of SFC in SF2 is higher than CB. At 40°C both

CB and CBE melted completely. In the study of Vereecken et al. (2009), the higher SFC at high

temperatures and lower SFC at low temperatures was attributed to the increased amount of SSS

TAGs in the fat sample.

Figure 30: Non-isothermal (non-tempered and tempered) SFC curve of the product, purified product, SF2 (CBE) and CB as measured by pNMR.

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40 50 60

SFC

(%

)

temperature (°C)

product tempered

product

purified product tempered

purified product

SF2 tempered

SF2

CB tempered

CB


5. Chemical composition CB/ CBE mixtures First, the FA profile of CB and CBE was determined using GC. Secondly, the produced CBE was mixed

with CB in different ratios (0, 20, 40, 60, 80 and 100% CBE). The TAG composition was determined of

each mixture.

5.1 FA profile of CB and CBE The results are shown in table 16. P, O and St are the three major fatty acids in the samples but also

considerable amounts of L (C18:2) and A (C20:0) were present. The amount of P and O in CBE was

significantly (p<0.05) higher than in CB. CBE contained significantly (p<0.05) less St compared to CB.

According to Khumalo et al. (2002), CB contains typically 34% St, 26% P and 35% O. The amount of P,

O and St in the analyzed CB was very close to the values mentioned in literature.

Table 16: FA composition of CB and CBE. The results are the average of two repetitions.

CB CBE

C16:0 (P) 27.66 ±0.13 28.62 ±0.07

C18:0 (St) 35.02 ±0.18 33.13 ±0.07

C18:1c (O) 32.48 ±0.03 34.62 ±0.05

C18:2 (L) 2.71 ±0.20 2.10 ±0.12

C20:0 (A) 0.87 ±0.12 0.37 ±0.01

∑others 0.96 ±0.01 1.04 ±0.08

5.2 TAG composition The TAG composition of the CB/ CBE mixtures was analyzed with HPLC using ACN and DCM as mobile

phases according to the procedure described by Rombaut et al. (2009). The amount of the three

main TAGs (POP, POSt and StOSt) is given in figure 31 (A). The amount of POP, POSt and StOSt in CB

was respectively 20.82, 41.57 and 33.89%. For the produced CBE, these amounts were respectively

21.52, 36.91 and 27.71%.

According to Lipp et al. (2001), a CBE contains generally a lower amount of POSt, a larger amount of

POP but a similar amount of StOSt. For the produced CBE, this statement was only confirmed in the

case of POSt. The amount of POP was similar and the amount of StOSt was significantly lower in the

produced CBE.

The amount of StOSt in CB was significantly (p<0.05) higher compared to the mixtures with CBE. The

total amount of the three main TAGs was significantly (p<0.05) higher in CB compared to the mixture

when 80% or more CBE was added.


The amount of SSS and SUU TAGs in each sample is presented in figure 31 (B). The reference CB

contained 1.64% and 1.56% SUU and SSS TAGs, respectively while these amounts for CBE were 8.42%

and 4.61%. When more CBE was added, the amount of SSS and SUU in the sample increased, having

opposite effects on the melting behavior of the samples. A higher amount of SSS will increase the

melting point while a higher amount of SUU will decrease the melting point.

Figure 31: Percentage of POP, POSt and StOSt (A) and SUU, SSS (B) TAGs in the CB/ CBE mixtures.

Figure 32 gives a ternary phase diagram. The area marked with the red line shows the ratios of POP,

POSt and StOSt which still give the same tempering characteristics as CB. This means that exactly the

same ratios of POP, POSt and StOSt as CB are not necessary (Timms, 2003; Padley et al., 1981). When

plotting the ratio of POP, POSt and StOSt of the produced CBE in this ternary phase diagram, it was

noticed that it fell into the marked area and not so far from CB. This means that the produced CBE

will have the same tempering characteristics as CB (Padley et al., 1981).

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

TAG

(%

)

CBE added (%)

StOSt

POSt

POP

0

1

2

3

4

5

6

7

8

9

10

0 20 40 60 80 100

TAG

(%

)

CBE added (%)

SUU

SSS

A B


Figure 32: POP/POSt/StOSt ternary diagram showing the position of CB, vegetable fats used as CBE and the

enzymatically produced CBE (Padley et al., 1981; Smith, 2001).

Silver ion HPLC was used to determine the symmetric and asymmetric TAGs present in CB/ CBE

mixtures. Stereospecific analysis of triglycerides is important because it influences the physical

behavior (melting properties, crystallization behavior and polymorphism) of fats and oils.

The results of symmetrical and asymmetrical TAGs for different CB/ CBE mixtures are shown in figure

33. There were no SSU TAGs detected in CB while in CBE there was 2.11% of SSU TAGs present. When

the amount of CBE in the mixture was raised, the amount of SSS, SSU and SUU TAGs increased. The

opposite was noticed for the amount of SUS TAGs, which decreased when the amount of CBE was

increased. High amounts of asymmetrical TAGs in the produced CBE are undesirable. According to

Vereecken et al. (2010), asymmetrical TAGs result in a slow crystallization of the fat as they have

lower melting points compared to the analogue symmetrical TAGs.

SSU TAGs are the result of acyl migration. Several factors cause acyl migration during the reaction.

These factors are an increase in reaction temperature, reaction time, water content and type of the

enzyme.


Figure 33: Percentage of SSS, SUS, SSU and SUU TAGs in the CB/ CBE mixtures; results obtained by silver ion

HPLC.

6. Physical characterization

In the next part, the physical characteristics of the CB/ CBE mixtures will be described and compared.

It is fundamental to have some insight in the different processes involved in fat crystallization to be

able to evaluate the final structure, functionality and quality of the CBE. Three levels in the structure

of the fat can be identified: a nano-, micro- and macro-scale. The primary crystallization (nano scale)

is characterized by nucleation and crystal growth and is followed by aggregation into clusters and

network formation (microscopic scale) (Dewettinck & Depypere, 2010). For instance, the snap of

chocolate depends strongly on the macroscopic properties of the CB fat network (Marangoni &

Narine, 1999).

First the non-isothermal and isothermal crystallization and melting behavior was described using DSC

and pNMR. In the second part, polarized light microscopy (PLM) was used to visualize the isothermal

crystallization.

The melting behavior of the produced CBE was compared to the melting behavior of CB, and

mixtures of CB with CBE in different ratios (0, 20, 40, 60, 80 and 100% CBE) were analyzed. The

results of DSC were compared with the ones obtained by pNMR and PLM.

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

TAG

(%

)

CBE added (%)

SUU

SSU

SUS

SSS


6.1 Non-isothermal crystallization and melting behavior


Figure 34 shows the DSC graphs of the mixtures of CB and CBE in different ratios. The values of the

different parameters after integration of the peaks are given in table 17.

Figure 34: Non-isothermal crystallization and melting profile of CB and the mixtures with CBE as measured by

DSC.

Table 17: Parameters Tonset (°C), Tpeak (°C), melting heat (J/g) and width at half height (°C) of DSC melting profile (non-isothermal) for mixtures of CB and CBE.

Sample Tonset (°C) Tpeak (°C) Melting heat (J/g) Width at half height (°C)

CB 14.05 ± 0.04 21.45 ± 0.15 77.76 ± 5.37 6.59 ± 0.08

20% CBE 14.71 ± 0.06 21.15 ± 0.23 76.97 ± 1.17 6.56 ± 0.08

40% CBE 15.06 ± 0.27 20.49 ± 0.16 77.31 ± 1,40 5.86 ± 0.49

60% CBE 12.88 ± 0.01 20.57 ± 0.05 76.48 ± 0,77 6.52 ± 0.01

80% CBE 12.55 ± 0.35 20.09 ± 0.16 73.41 ± 0.35 6.59 ± 1.05

100% CBE 13.09 ± 0.22 20.42 ± 0.16 69.36 ± 1.85 7.56 ± 0.21

When comparing the crystallization peaks of CB with the mixtures containing CBE, one can notice a

shoulder appearing before the main crystallization peak (indicated by A on figure 34). CB had no

shoulder in its crystallization curve. This was due to a higher amount of SSS TAGs present in the CBE.

CB contained 1.56% of SSS TAGs compared to 4.61% in the produced CBE. Especially the amount of

PPP differed a lot between CB (0.91%) and CBE (1.96%). According to Corrêa Basso et al. (2010), high

amounts of PPP increase the crystallization rate that could explain the appearance of this shoulder.

A

B


Adding less CBE meant there were less SSS TAGs present in the mixture and this resulted in a smaller

shoulder. When this shoulder was smaller, the main crystallization started earlier and the onset

temperature was higher. This can be seen in table 17 where Tonset for CB and CBE were respectively

14.05°C and 13.09°C.

Generally, the onset temperature decreased when more CBE was added. This is probably due to the

higher amount of low melting fraction (UUU and SUU TAGs) present in the CBE. Another explanation

is the higher amount of SSU TAGs present when more CBE was added. Vereecken et al. (2010) stated

that, for instance, PPO (34.5°C) had a lower melting point than POP (36°C) resulting in a faster

melting which could explain the lower Tonset when more CBE was added. Also the peak temperature

was significantly lower compared to CB when more CBE was added. This is due to a larger amount of

SUU TAGs present in the CBE.

After the main melting peak, a smaller melting peak occurred for the mixtures (indicated by B on

figure 34). This is due to a considerable amount of SSS TAGs in the CBE, this was also noticed by

Cebula & Smith (1992).

The shift of the melting peak towards lower temperatures was due to a relative higher amount of

SUU TAGs present in the sample which have lower melting points. In figure 34, a small shift of the

melting peak was noticed when more CBE was added to the CB.

6.1.2 Solid fat content as measured by pNMR

The particular melting behavior of chocolate is due to CB. Through pNMR, the SFC is determined with

the tempered and non-tempered procedure, giving information about the melting behavior of each

sample. The melting curve of CB is compared to those of mixtures of CBE with CB in different ratios.

In figure 35 (A) the results of the pNMR non-isothermal method (non-tempered) are shown when the

CBE was mixed in different ratios with CB. All mixtures had lower SFC than CB up to 30°C. The melting

profile of all mixtures was very similar to that of the CB but, at the same temperature, when the

amount of CBE increased, the SFC decreased. The mixtures were completely melted between 35 and

40°C which was similar to CB. Vereecken et al. (2010) noticed a small decrease in SFC when more SSU

TAGs were present.

During tempering of the samples, the fat crystals are transformed to the stable β-polymorphic form.

Around the tempering temperature (26°C), the unstable fat crystals (α) will melt and the more stable

crystals of polymorphic form βV remain. This polymorph causes the narrow melting profile of CB.

When other polymorphic crystals are present in the sample, a broader melting profile is obtained

(Timms, 2003). The SFC of the tempered samples is given in figure 35 (B).


Figure 35: Non-isothermal non-tempered (A) and tempered (B) SFC curve of the CBE-CB mixtures as

measured by pNMR.

In figure 36 a comparison is made between tempered and non-tempered CB and CBE.

According to Timms (2003), below the tempering temperature, a tempered sample has a lower SFC

compared to a non-tempered sample. Above this temperature, the SFC of a tempered sample is

higher. The tempering temperature in this research was 26°C. When looking at the results, one can

notice that only up to 20°C, the SFC of the tempered sample was lower than the non-tempered

sample of CB.

The same conclusion can be made for the CBE but the difference between tempered and non-

tempered CBE was not as big as for tempered and non-tempered CB. Beyond 20°C, the tempered

sample had indeed a higher SFC than the non-tempered sample resulting in a higher melting point.

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40

SFC

(%

)

Temperature (°C)

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40

SFC

(%

)

Temperature (°C)

A B


Figure 36: Non-isothermal SFC curve: comparison of tempered and non-tempered CB and pure CBE as

measured by pNMR.

Different zones can be distinguished in the SFC curve of the tempered samples. The SFC below room

temperature (25°C) indicates the hardness of the fat. Between 25°C and 35°C, the SFC gives an

indication of the heat resistance and if the fat has a high SFC at temperatures above body

temperature (37°C), a waxy mouth feel of the product can be noticed (Talbot, 2009b; Torbica et al.,

2006). The results of these zones of CB and the mixtures with produced CBE are given in figure 37.

As it is clear from figure (A), up to the temperature of 15°C, there was no difference in hardness for

the CB and the mixtures containing the produced CBE. At higher temperatures, the hardness

decreased when the amount of CBE increased. Regarding the heat resistance (B), when more CBE

was added, a lower heat resistance was obtained. The lower heat resistance was probably due to a

relative higher amount of SUU and SSU TAGs present in the produced CBE. SUU and SSU TAGs have

lower melting points and result in a lower SFC at the same temperature compared to CB. Finally,

from 35°C (C), the waxiness increased when more CBE was used in the mixture. The higher amount of

SFC at 35°C in the samples containing CBE, was due to the higher amount of SSS TAGs which melt at

higher temperature.

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40

SFC

(%

)

Temperature (°C)

CB tempered

CB non-tempered

CBE tempered

CBE non-tempered


Figure 37: SFC melting curves indicating the hardness (A), heat resistance (B) and waxiness (C) of CB and

mixtures with CBE (Depoortere, 2011).

6.1.3 Isothermal diagram

In an isothermal diagram, the SFC of the mixtures of CB and CBE are shown as function of the ratio of

CBE in the mixture and the temperature. According to Ciftci et al. (2010), full compatible samples

should result in isotherms that are horizontal lines between the SFC of pure CB and the SFC of pure

CBE at a given temperature.

Figure 38 gives the SFC of the tempered mixtures of CB with CBE measured at different

temperatures. Due to the tempering of the samples, the β-polymorphic form of the fat crystals was

obtained.

It seems that when the temperature was increased, the produced CBE became less compatible with

CB. At a temperature between 20 to 30°C, a higher amount of CBE resulted in a lower SFC. This is due

to the higher amount of SUU TAGs present in the CBE. At 35°C, the SFC is higher when more CBE was

added to the CB because of the higher amount of SSS TAGs in the CBE. This last characteristic, and in

combination with a similar SFC at lower temperatures, is typical for cocoa butter improvers (CBI)

(Pontillon, 1998).

0

20

40

60

80

100

5 10 15 20 25

SFC

(%

)

Temperature (°C)

Hardness

0

20

40

60

80

25 30 35

SFC

(%

)

Temperature (°C)

Heat resistance

0

10

20

30

40

50

30 35 40

SFC

(%

)

Temperature (°C)

Waxiness CB

20% CBE

40% CBE

60% CBE

80% CBE

100% CBE

A

B

C


Figure 38: Isothermal diagram of the mixtures of CBE and CB.

6.2 Isothermal crystallization For the isothermal crystallization, samples were held at 20°C. Measurements were performed using

DSC and pNMR.

6.2.1 Isothermal crystallization as measured by DSC

The crystallization was performed at 20°C. At higher temperatures, the crystallization would be too

slow and at lower temperatures the fat would already crystallize during cooling up to the isothermal

temperature, making it difficult to study the isothermal crystallization.

At 20°C, a two-step crystallization process was noticed which is presented in figure 39. First α-crystals

are formed, followed by a transformation to the β’-polymorphic form during the second step (Toro-

Vazquez et al., 2005). The duration of the experiment was not long enough for the β’-polymorphs to

transform into the more stable β-polymorphs. According to Foubert (2003), this polymorphic form

can only be obtained after at least one week of storage at room temperature. The peak of the α-

polymorph cannot be integrated since it overlaps with the peak at the beginning of the isothermal

method. Therefore, only the peak of the β’-polymorphic form will be considered during integration of

the results.

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

SFC

(%

)

CBE (%)

5°C

10°C

15°C

20°C

25°C

30°C

35°C

40°C


Figure 39: Isothermal crystallization of CB at 20°C as measured by DSC.

Figure 40 gives the DSC graph of mixtures of CB and CBE using the isothermal method at 20°C. From

this figure it is clear that the crystallization at 20°C slowed down when more CBE was added. The

peak maximum will be lower when more CBE was added. Also, adding more CBE resulted in

increased tailing of the crystallization peak. This means that more time was needed for these

samples to crystallize completely. The same flat heat-flow curves, due to the very slow crystallization

of CBE, were noticed by Kerti (2001). Adding CBE also caused the crystallization to start later than

was the case for the CB.

All of these differences can be explained by the higher amount of UUU and SUU TAGs in the

produced CBE. In CB, only 1.64% of POO and StOO were present while the produced CBE still

contained 8.42% of OOO, POO and StOO. Besides, the amount of SSU TAGs may slow down the

crystallization. Especially OOO had an influence on the crystallization rate. Foubert et al. (2004b)

noticed that the β’ melting point for POO (2.5°C) and StOO (8.6°C) caused these SUU TAGs not to

crystallize at 20°C. Therefore, a slow crystallization was noticed when more SUU and SSU TAGs were

present (Vereecken et al., 2010). However, because of the high amount of SSS TAGs, co-

crystallization can occur which can make these SUU TAGs to crystallize.

α-crystallization peak β’-crystallization peak


Figure 40: Isothermal crystallization at 20°C of the different ratios of CB and CBE.

Foubert et al. (2002) developed a model which describes the kinetics of the isothermal crystallization

of fats. The kinetics of crystallization of fat is important when trying to obtain desired product

characteristics (Foubert et al., 2008). Foubert et al. (2004a) demonstrated that, compared to other

mathematical models which describe isothermal crystallization, the Foubert model shows a better fit

to the data. Moreover, the Foubert model offers flexibility towards asymmetric DSC curves. In the

Foubert equation, four parameters are important. These four parameters and the differential

equation are (Foubert, 2003):

tind (h): the time needed to obtain x% crystallization and x is chosen to be 1.

K (h-1): the rate constant

aF (J/g): the maximum amount of crystallization

n (-): the order of the reverse reaction


It is important to notice that the Foubert model was developed for single-step crystallization

processes, only the major crystallization peak is taken into account. Therefore, in this research only

the parameters of the β’-crystallization were compared (Calliauw, 2008a). The model was fitted to

the data, and the start- and endpoint of the integration was determined using the calculation

algorithm as developed by Foubert (2003). The value of parameter n is fixed at 6 in order to

determine the changes in K. This value for β’-crystallization was also found by Foubert et al. (2006)

when analyzing the two-step crystallization process of CB at 20°C. Table 18 gives the Foubert

parameters for the CB and mixtures with produced CBE.

The amount of equilibrium solid fat decreased when more CBE was added to the mixture. aF of each

mixture containing CBE, differed significantly (p<0.05) from the aF of CB. The higher aF of CB can be

explained by the higher amount of POSt (41.57%) compared to the CBE (39.91%). According to

Calliauw (2008a), the amount of POSt in a fat possitively correlates with the aF. On the other hand;

DAGs and FFA lower the aF (Calliauw et al., 2008b). Since no DAGs were detected in CBE and FFA

were removed after SPD, this could not be the reason of the decrease in aF in the CBE. aF decreased

because of the higher amount of SUU TAGs present (Foubert et al., 2004b)

tind indicates the time that was necessary to start the transformation from the α-crystals into the β’-

polymorphic form. One can notice that when more CBE was added, the tind increased, resulting in a

later transformation start. According to Chaiseri & Dimick (1995), the fat will be softer (lower SFC)

when more SUU TAGs (POO and StOO) are present, resulting in a longer induction time. Also, a

higher amount of FFA will increase the tind. The produced CBE contained 8.42% SUU compared to

1.56% in CB. The addition of more CBE resulted in a longer induction time due to the higher amount

of SUU TAGs. The lower tind of CB can be explained by the higher amount of POSt compared to CBE.

Parameter K decreased significantly (p<0.05), compared to CB, when more CBE was added. This is

probably due to the higher amount of SUU TAGs present in the CBE which decreased the rate

constant. Also a higher amount of DAGs will lower the value of K but this was not the case in this

research because of the absence of DAGs in the CBE.


Foubert et al. (2004b) stated that all parameters, except K, are influenced by the ratio of saturated

(Sat FA) to unsaturated FA (Unsat FA) and the ratio of SUS to SUU TAGs. When the amount of Unsat

FA and SUU TAGs increases, aF will decrease and tind will increase. The results of these ratios for the

CB/ CBE mixtures are given in figure 41. The Sat FA are the sum of P, St and A; the Unsat FA are the

sum of O and L as analyzed by GC. The ratio of SUS to SUU TAGs and the value for aF are indicated on

the primary y-axis on the left of the graph. The ratio of Sat FA to Unsat FA and the value of tind are

indicated on the secondary y-axis on the right of the graph. When both ratios decreased, a decrease

in aF and an increase in tind was noticed. This was in accordance with the results of Foubert et al.

(2004b).

Table 18: Parameters aF (J/g), tind (h) and K (h-1

) of the Foubert model for mixtures of CB and CBE.

Sample aF (J/g) tind (h) K (h-1)

CB 72.54 ± 2.06 0.48 ± 0.78 4.63 ± 0.01

20% CBE 67.10 ± 0.85 0.49 ± 0.00 3.14 ± 0.06

40% CBE 62.37 ± 0.03 0.51 ± 0.06 2.21 ± 0.05

60% CBE 52.82 ± 0.78 0.53 ± 0.06 1.63 ± 0,10

80% CBE 50.16 ± 2.57 0.76 ± 0.57 1.31 ± 0.27

100% CBE 49.39± 0.68 1.45 ± 0.04 0.72 ± 0.16

Figure 41: Influence of Sat FA to Unsat FA and SUS to SUU TAGs ratios on aF and tind.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100

Sat

FA/U

nsa

t FA

an

d t

ind

(h

)

SUS/

SUU

an

d a

F (J

/g)

CBE added (%)

SUS/SUU

aF

Sat FA/Unsat FA

tind


6.2.2 Isothermal crystallization as measured by pNMR

In figure 42 the isothermal crystallization of the mixtures CB/CBE at 20°C followed by pNMR is given.

In this figure, the two-step crystallization was observed. The insert of the first 50 min (figure 42),

showed the formation of the α-crystals and the beginning of the transformation into the β’-

polymorphic form as was also noticed by Dewettinck et al. (2004). It can be concluded that the

nucleation of CB started later. In other words; the crystallization (of α-crystals) started sooner when

the amount of CBE in the mixture was increased. This was due to the higher amount of SSS TAGs in

the CBE. Possibly, the higher amount of FFA (1.32%) in the CB resulted in the slower nucleation. The

opposite was noticed for the second crystallization step in which α-crystals are transformed into the

β’-polymorph. When less CBE was added to the mixture, the increase in this second crystallization

step was steeper. Or; when more CBE was added, the transformation of α- to β’-crystals occurred

slower. This was in accordance with the results obtained by isothermal DSC and can be explained by

the higher amount of SUU TAGs present in the CBE. Ribeiro et al. (2012) stated that the longer

induction period before β’ crystallization was due to a higher amount of SUU TAGs.

After 230 min of measuring the SFC, CB reached a SFC content of 73% while pure CBE only reached a

SFC of 45%. Consequently, it can be concluded that the crystallization was slower when more CBE

was added to the mixture.


Figure 42: Isothermal crystallization at 20°C of CB and mixtures with CBE as measured by pNMR.

0

10

20

30

40

50

60

70

80

0 50 100 150 200 250

SFC

(%

)

Time (min)

CB

20%

40%

60%

80%

100%

0

5

10

15

20

25

30

35

40

45

0 10 20 30 40 50

SFC

(%

)

Time (min)

CB

20%

40%

60%

80%

100%


6.3 Isothermal crystallization as visualized by PLM Polarized light microscopy (PLM) was used to visualize the isothermal crystallization at 20°C of the

mixtures of CB and CBE into crystals, crystal clusters and the formation of crystal networks. Firstly,

the beginning of isothermal crystallization was recorded. Secondly, the crystallization at 20°C was

traced during a follow-up of 6 weeks.

6.3.1 Start of isothermal crystallization at 20°C

Pictures of the crystal formation were taken after 1, 10, 30, 60 and 90 min of isothermal

crystallization at 20°C. In order to determine the relationship between microstructure and

polymorphism of CB, the temperature of 20°C is crucial. Below 20°C and depending on the duration

of the crystallization, the CB can have a granular morphology characteristic for the β’-polymorph

while higher temperatures can promote the crystallization into clusters with high polymorphic

stability (Ribeiro et al., 2012). The result after 1 min is given in figure 45 in appendix. The black

background was the liquid fat, the crystals were polarized and they appeared as the white spots in

the picture.

The pictures show that CB (a) almost didn’t show any crystal after 1 min. When more CBE was added,

more crystals in a granular appearance were noticed, corresponding to the formation of α-crystals.

This means that first the α-crystals were formed. From the isothermal pNMR results, it was clear that

the nucleation started earlier when more CBE was added. Therefore, in the samples containing more

CBE, crystals were noticed earlier compared to CB where the nucleation started later. The

explanation is the higher amount of PPP present in the CBE compared to CB. Vereecken et al. (2009)

noticed that a higher amount of PPP resulted into bigger granular crystals. This can be seen in the

picture where adding more CBE resulted into slightly larger granular crystals. Campos et al. (2010)

noticed that a higher amount of StStSt present, decreased the onset time of crystallization. The

produced CBE contained more StStSt compared to CB, which resulted into a faster formation of α-

crystals.

The result after 60 min is given in figure 43. Compared to the results after 1 min, the granular

appearance became more clear and denser although the difference was not that clear. The same

conclusion can be made when more CBE was added, the α-crystals were formed faster and in a larger

amount.


Figure 43: Isothermal crystallization at 20°C after 60 min as visualized by PLM: 0 (a), 20 (b), 40 (c), 60 (d), 80

(e) and 100% (f) CBE.

After the isothermal crystallization, the samples were kept at 20°C for 6 weeks. Pictures were taken

on a regular base to evaluate the crystal formation.

6.3.2 6 week follow-up

During 6 weeks, the samples were stored at 20°C. Every week, a picture of each sample was taken to

analyze the progress in crystal formation. Figure 44 gives the results after 24h, 2, 4 and 6 weeks of

isothermal crystallization. The results after 1 week, 3 weeks and 5 weeks are given in figure 46 in

appendix.

a b

c d

e f


After 24h, a denser network of small granular crystals was formed. Also CB formed a dense granular

network of crystals. The second step in the crystallization process was noticed: the formation of α-

into β’-crystals. At 24h, this process also occurred for CB.

From the results of isothermal DSC and pNMR, it was stated that the transformation of α- into β’-

crystals occurred faster for CB. This was also noticed with PLM where a denser granular crystal

network was seen compared to the mixtures containing CBE. When more CBE was added, less crystal

formation was noticed. This was probably due to the higher amount of SUU and SSU TAGs present in

the produced CBE. These TAGs slow down the crystallization process as was stated after isothermal

DSC. The delay in polymorphic transition due to a higher amount of StStSt present in the CBE, was

explained by Campos et al. (2010).

From week 2, bigger, featherlike crystals, specific for β-crystals, were formed when 40% or less CBE

was added. Also the formation of a large amount of small crystals was noticed when 60% or more

CBE was used. After week 4, a different form of crystals was noticed when 40% or more CBE was

added compared to the CB crystals. 100% CBE still crystallized into a lot of very small crystals,

indicating that the crystallization of CBE was still going on. In the study of Vereecken et al. (2009), the

crystal network of fat samples, higher in PPP, only became denser after one month of storage at

20°C. Therefore, they stated that a higher amount of SSS TAGs lead to smaller crystals. In the

meantime, CB and 20% CBE formed large and dense crystal aggregates.

The pictures after week 6 showed the featherlike structure of the crystals when 40% or less CBE was

used. Looking at the crystals of 40% CBE, one could notice two different morphologies in the crystal

structure. The center of the big crystals had a granular structure while on the outside, featherlike

crystals surrounded the granular crystals. When 60% CBE was added, also two different forms in

which the crystals aggregated were noticed; a more featherlike part and a spiral like structure. The

crystals of the 80% CBE mixture started to grow more but in comparison with the previous samples,

there were still a lot of small crystal aggregates. The pure CBE still contained the granular and dense

structure of very small crystals.

In conclusion; crystal formation was faster for CB resulting in larger crystals aggregates. Each week,

the CB crystal network became larger and denser compared to the mixtures where CBE was added.

When more CBE was used, less crystal aggregates were formed and they were smaller than the CB

crystal network. This was another indication of the very slow crystallization of the produced CBE.


Figure 44: Isothermal crystallization at 20°C as visualized by PLM for CB, 20, 40, 60, 80 and 100% CBE. The

microstructure is given after 24h, 2 weeks, 4 weeks and 6weeks.


General conclusions The purpose of this study was to produce a CBE by enzymatic acidolysis starting from the cheap and

commercial available HOSO, and to compare the produced CBE with CB, chemically and physically.

In the first step, the quality of HOSO and its chemical composition, in order to be used as a source in

enzymatic acidolysis reactions, was analyzed. The oil was of good quality and contained a high

amount of OOO (62.09%). Having a high amount of TAGs with O at the sn-2 position, made this oil an

ideal source for CBE using the sn-1,3 specific RM IM and the FAM of P and St.

Secondly, the enzymatic acidolysis of HOSO with FAM was performed and the optimized value of

each parameter as obtained by RSM was: reaction time (8h), reaction temperature (65°C), water

content (1%), enzyme load (8.54%) and the substrate ratio (7.99 mol FAM: 1 mol HOSO).

Some attempts were made in order to improve the yield by adding glycerol and Novozyme 435 to the

interesterification reaction with HOSO. The result was a reduction of FFA from 65% to 9% when only

glycerol was added in a molar ratio of 1:4 (mol substrate: mol glycerol). However, the amount of DAG

increased up to 67% and the amount of desired TAGs (POP, POSt and StOSt) decreased from 20% to

13.45%. Ideally, a maximum of 2 mol glycerol can be added to reduce the FFA to approximately 36%

and 35% of DAG. Adding glycerol and the non-specific enzyme together, resulted into the same

trends of decreasing FFAs, decreasing TAGs and increasing amount of DAGs. Since it is more difficult

to remove DAGs compared to FFAs, it was concluded not to include glycerol and a non-specific

enzyme in the reaction.

The purification of the interesterified HOSO by SPD resulted in only 0.32% of FFA. Through solvent

fractionation, the amount of SUU and SSS TAGs was reduced to 8.42% and 4.61%, respectively.

Compared to CB, 1.64% SUU and 1.56% SSS, these amounts were high. The CBE contained a similar

amount of POP, and lower amounts of POSt and StOSt compared to CB. Also the FA profile was

similar to that of CB. Therefore, it can be concluded that the produced CBE will have the same

tempering characteristics as CB.

In the last part of the research, the physical characteristics of the produced CBE, when mixed in

different ratios with CB, were determined. From the results of non-isothermal DSC, a shoulder before

the main crystallization peak was noticed. This peak was due to the higher amount of SSS TAGs

present in the CBE.

The solid fat curve as measured by pNMR was similar for CBE as to CB at temperatures between 20

and 30°C. However, at lower temperatures, CBE had a lower SFC due to the higher amount of SUU

TAGs and at increasing temperatures, a higher SFC was obtained because of the larger amount of SSS

TAGs in the CBE which may result in a higher waxiness from 35°C.


The two-step crystallization process was visualized by isothermal DSC and pNMR at 20°C. Due to the

higher amount of SSS TAGs present in the CBE, the nucleation into α-crystals started earlier for the

mixtures containing CBE than for CB. tind, indicating the time that was necessary to start the

transformation from the α-crystals into the β’-polymorphic form, increased when more CBE was

added to the mixture. On the other hand, the rate constant (K) decreased when more CBE was added.

These observations lead to a slow crystallization behavior of the produced CBE.

The isothermal crystallization at 20°C was also visualized by PLM during a period of six weeks. During

these weeks, it was clear that the CBE formed very quickly granular crystals (α-crystals) but this

granular network only changed very slowly into aggregates and β-crystals. The CB crystals

transformed fast into big and featherlike crystal aggregates compared to the CBE which still had very

small, granular crystals after five weeks. A different morphology of the crystals was noticed when

CBE was added to CB compared to pure CB. Adding CBE resulted in less dense crystal networks in the

shape of a spiral.

By using enzymatic acidolysis, it is possible to produce a CBE from HOSO, P and S with a chemical

composition that is very close to that of CB. However, the physical characteristics of the CBE were

different, resulting into a very slow crystallization process compared to CB. Therefore, according to

the results, when using this CBE in CB, small amounts (maximum 20%) could be applied with limited

changes to the typical physical properties of CB.


Further research For the production process of CBE, it would be beneficial if methods were found to minimize the

amount of FFA left in the esterified product. One possibility is the use of ultrafiltration which is a

membrane technology. With the removal of water during the enzymatic acidolysis reaction, the yield

of the reaction can be increased. Possible methods to remove the water are adding salt hydrates or

applying pervaporation (Ghandi et al., 2000).

For further research concerning the produced CBE, it might be interesting to use the CBE in different

ratios into chocolate. It can be tested if the pure CBE has a big effect on rheological and textural

properties of chocolate.

The CBE produced by enzymatic acidolysis can be compared to commercial available CBEs in order to

study if the produced CBE shows better chemical and physical compatibility with CB. Also the major

differences between commercial available CBEs and enzymatic produced CBE can be evaluated.


References 1. Abigor R.D., Marmer W.N., Foglia T.A., Jones K.C., Diciccio R.J., Ashby R. & Uadia R.O., 2003.

Production of cocoa butter-like fats by the lipase-catalyzed interesterification of palm oil and

hydrogenated soybean oil. Journal of American Oil Chemists’ Society, 80 (12), 1193-1196.

2. Bockisch M., 1998. Fats and Oils Handbook. Illinois, AOCS Press, 838 p.

3. Calliauw G., 2008a. Molecular interactions affecting the phase composition during dry

fractionation of palm olein. PhD thesis, Ghent University, Belgium, 275p.

4. Calliauw G., Vila Ayala J., Gibon V., Wouters J., De Greyt W., Foubert I. & Dewettinck K.,

2008b. Models for FFA-removal and changes in phase behaviour of cocoa butter by packed

column steam refining. Journal of Food Engineering, 89, 274-284.

5. Campos R., Ollivon M. & Marangoni A.G., 2010. Molecular composition Dynamics and

Structure of Cocoa Butter. Crystal Growth & Design, 10 (1), 205-217.

6. Cebula D.J. & Smith K.W., 1992. Differential scanning calorimetry of confectionery fats. Part II:

Effects of blends and minor components. Journal of American Oil Chemists’ Society, 66,

1771-1776.

7. Chaiseri S. & Dimick P.S., 1995. Dynamic crystallization of cocoa butter. I. Characterization of

simple lipids in rapid- and slow-nucleating cocoa butters and their seed crystals. Journal of

American Oil Chemists’ Society, 72, 1491-1496.

8. Chang M.K., Abraham G. & John V.T., 1990. Production of Cocoa Butter-Like Fat from

Interesterification of Vegetable Oils. Journal of American Oil Chemists’ Society, 67 (11), 832-

834.

9. Chong C.N., Hoh Y.M. & Wang C.W., 1992. Fractionation Procedures for Obtaining Cocoa

Butter-Like Fat from Enzymatically Interesterified palm Olein. Journal of American Oil

Chemists’ Society, 69 (2), 137-140.

10. Chopra R., Reddy Yella Reddy S. & Samboiah K., 2008. Structured lipids from rice bran oil and

stearic acid using immobilized lipase from Rhizomucor miehei. European Journal of Lipid

Science and Technology, 110, 32-39.

11. Ciftci O.N., Fadiloglu S. & Gögüs F., 2009. Conversion of olive pomace oil to cocoa butter-like

fat in a packed-bed enzyme reactor. Bioresource Technology, 100, 324-329.

12. Ciftci O.N., Fadiloglu S., Kowalski B. & Gögüs F., 2008. Synthesis of cocoa butter

triacylglycerols using a model acidolysis system. Grasas y aceites, 59 (4), 316-320.


13. Ciftci O.N., Gögüs F. & Fadiloglu S., 2010. Performance of a Cocoa Butter-Like Fat

Enzymatically Produced from Olive Pomace Oil as a Partial Cocoa Butter Replacer. Journal of

American Oil Chemists’ Society, 87, 1013-1018.

14. Corrêa Basso R., Badan Ribeiro A.P., Masuchi M.H., Gioielli L.A., Guaraldo Goncalves L.A.,

Oliveira dos Santos A., Pavie Cardoso L. & Grimaldi R., 2010. Tripalmitin and

monoacylglycerols as modifiers in the crystallisation of palm oil. Food Chemistry, 122, 1185-

1192.

15. De Clercq N., 2011. Changing the functionality of cocoa butter. PhD Thesis, Ghent University,

Belgium, 220 p.

16. Depoortere L., 2011. The use and applicability of cocoa butter equivalents (CBEs) in chocolate

products. Master thesis, Ghent University, Belgium, 82p.

17. Dewettinck K. & Depypere F., 2010. Formulering en structurering van levensmiddelen. Cursus.

Universiteit Gent, 194 p.

18. Dewettinck K., Foubert I., Basiura M. & Goderis B., 2004. Phase behavior of cocoa butter in a

two-step isothermal crystallization. Crystal Growth & Design, 4 (6), 1295-1302.

19. Elibal B., Funda Suzen H., Ayse Aksoy H., Ustun G. & Tuter M., 2011. Production of structured

lipids containing conjugated linolenic acid: optimisation by response surface methodology.

International Journal of Food Science & Technology, 46, 1422-1427.

20. Esteban L., Jimenez M.J., Hita E., Gonzalez P.A., Martin L. & Robles A., 2011. Production of

structured triacylglycerols rich in palmitic acid at sn-2 position and oleic acid at sn-1,3

positions as human milk fat substitutes by enzymatic acidolysis. Biochemical Engineering

Journal, 54 (1), 62-69.

21. EU Directive 2000/36/EC of the European Parliament and of the Council of 23 June 2000

relating to cocoa and chocolate products intended for human consumption. Official Journal

of the European Communities, L197, 19-25.

22. European patent specification: European patent 0 199 580 B1.

23. Fadiloglu S., Ciftci O.N. & Gögüs F., 2003. Reduction of Free Fatty Acid Content of Olive-

pomace Oil by Enzymatic Glycerolysis. Food Science and Technology International, 9 (1), 1-5.

24. Foubert I., 2003. Modelling isothermal cocoa butter crystallization: influence of temperature

and chemical composition. PhD thesis, Ghent University, Belgium, 263 p.


25. Foubert I., Dewettinck K., Janssen G. & Vanrolleghem P.A., 2006. Modelling two-step

isothermal fat crystallization. Journal of Food Engineering, 75, 551-559.

26. Foubert I., Fredrick E., Vereecken J., Sichien M. & Dewettinck K., 2008. Stop-and-return DSC

method to study fat crystallization. Thermochimica Acta, 471, 7-16.

27. Foubert I., Vanrolleghem P.A. & Dewettinck K., 2004a. Comparison of models to describe

crystallization kinetics of fats. Modelling and Simulation 2004, 333-337.

28. Foubert I., Vanrolleghem P.A., Thas O. & Dewettinck K., 2004b. Influence of chemical

composition on the isothermal cocoa butter crystallization. Journal of Food Science, 69 (9),

478-487.

29. Foubert I., Vanrolleghem P.A., Vanhoutte B. & Dewettinck K., 2002. Dynamic mathematica

model of the crystallization kinetics of fats. Food Research International, 35, 945-956.

30. Ghandi N.N., Patil N.S., Sawant S.B. & Joshi J.B., 2000. Lipase-Catalyzed Esterification. Catal.

Rev.- Sci. Eng., 42 (4), 439-480.

31. Gitlesen T., Svensson I., Adlercreutz P., Mattiasson B. & Nilsson J., 1995. High-oleic acid

rapeseed oil as starting material for the production of confectionary fats via lipase-catalysed

transesterification. Industrial Crops and Products, 4, 167-171.

32. Gray J.I., 1978. Measurement of Lipid Oxidation: A Review. Journal of American Oil Chemists’

Society, 55, 539-546.

33. Holm H.C. & Cowan D., 2008. The evolution of enzymatic interesterification in the oils and

fats industry. European Journal of Lipid Science and Technology, 110, 679-691.

34. Hoy C. & Xu X., 2001. Structured Triacylglycerols. In: Gunstone F.D. (ed.) Structured and

Modified Lipids. Marcel Dekker Inc., New York, Basel, 209-239.

35. Huyghebaert A. & Hendrickx H., 1971. Polymorphism of cocoa butter shown by differential

scanning calorimetry. Lebensmittel- Wissenschaft und -Technologie, 4, 59-63.

36. ICCO, International cocoa organization, 2012. ICCO monthly averages of daily prices.

http://www.icco.org/statistics/monthly.aspx (consulted 24/04/2012).

37. Kellens M., Gibon V., Hendrix M. & De Greyt W., 2007. Palm oil fractionation. European

Journal of Lipid Science and Technology, 109, 336-349.

38. Kerti K., 2001. Investigating isothermal DSC method to distinguish between cocoa butter and

cocoa butter alternatives. Journal of Thermal Analysis and Calorimetry, 63, 205-219.


39. Khumalo L.W., Majoko L., Read J.S. & Ncube I., 2002. Characterisation of some underutilised

vegetable oils and their evaluation as starting materials for lipase-catalysed production of

cocoa butter equivalents. Industrial Crops and Products, 16, 237-244.

40. Lin S.W. & Yoo C.K., 2009. Short-path distillation of palm olein and characterization of

products. European Journal of Lipid Science and Technology, 111, 142-147.

41. Lipp M. & Anklam E., 1998. Review of cocoa butter and alternative fats for use in chocolate –

Part A. Compositional data. Food Chemistry, 62 (1), 73-97.

42. Lipp M., Simoneau C., Ulberth F., Anklam E., Crews C., Brereton P., De Greyt W., Schwack W.

& Wiedmaier C., 2001. Composition of Genuine cocoa butter and cocoa butter equivalents.

Journal of food composition and analysis, 14, 399-408.

43. Macher M.B. & Holmqvist A., 2001. Triacylglycerol analysis of partially hydrogenated

vegetable oils by silver ion HPLC. Journal of Separation Science, 24 (3), 179-185.

44. Marangoni A.G. & Narine S.S., 1999. Microscopic and rheological studies of fat crystal

networks. Journal of Crystal Growth 198/199, 1315-1319.

45. Martin D., Reglero G. & Señoráns F.J., 2010. Oxidative stability of structured lipids. European

Journal of Lipid Science and Technology, 231, 635-653.

46. Martins P.F., Ito V.M., Batistella C.B. & Maciel M.R.W., 2006. Free fatty acid separation from

vegetable oil deodorizer distillate using molecular distillation process. Separation and

Purification Technology, 48, 78-84.

47. Melo Branco M.E., Bueno Campos P.R., Noso T.M., Alberici R.M., da Silva Cunha I.B., Costa

Simas R., Nogueira Eberlin M. & de Oliveira Carvalho P., 2011. Response surface modeling of

the production of structured lipids from soybean oil using Rhizomucor miehei lipase. Food

Chemistry, 127, 28-33.

48. Novozymes, 2011. Enzymatic Interesterification using Lipozyme TL IM in Laboratory Scale

Batch Reactors. 8 p.

49. Oh J.E., Lee K.W., Park H.K., Kim J.Y., Kwon K.I., Kim J.W., Kim H.R. & Kim I.H., 2009. Lipase-

catalyzed Acidolysis of Olive Oil with Capric Acid: Effect of Water Activity on Incorporation

and Acyl Migration. Journal of agricultural and food chemistry, 57 (19), 9280-9283.

50. O’Keefe S.F. & Dike O.A., 2010. Fat characterization. In: Nielsen S.S. (ed.) Food Analysis.

Springer New York, 239-260.


51. Pacheco C., Crapiste G.H. & Carrin M.E., 2010. Lipase-catalyzed acidolysis of sunflower oil:

Kinetic behavior. Journal of Food Engineering, 98 (4), 492-497.

52. Padley F.B., Paulussen C.N., Soeters C. & Tresser D. (Lever Brothers CO.), 1981. Chocolate

having defined hard fat. US Patent 4726322.

53. Palla C.A., Pacheco C. & Carrin M.E., 2012. Production of structured lipids by acidolysis with

immobilized Rhizomucor miehei lipases: Selection of suitable reaction conditions. Journal of

Molecular Catalysis B: Enzymatic, 76, 106-115.

54. Paquot C. & Hautfenne A., 1984. Standard Methods for the Analysis of Oils, Fats and

Derivatives. Blackwell Scientific Publications.

55. Pinyaphong P. & Phutrakul S., 2009. Modification of palm oil structure to cocoa butter

equivalent by Carica papaya lipase-catalyzed interesterification. World Academy of Science,

Engineering and Technology, 54, 536-540.

56. Podmore J., 1990. Controlling Quality In An Edible Oil Refinery. In: Erickson D.R. (ed.) Edible

Fats and Oils Processing: Basic Principles and Modern Practices. American Oil Chemists’

Society, 374-389.

57. Pontillon J., 1998. Traitement du cocoa; les semi-produits. In: Pontillon J. (ed.) Cacao et

chocolat: production, utilisation, caractéristiques. Paris, Lavoisier, 363-393.

58. Rajah K.K., 1996 . Fractionation of fat. In: Grandison A.S. & Lewis M.J. (eds.) Separation

processes in the food and biotechnology industries. Principles and applications. UK,

Woodhead Publishing Series in Food Science and Technology, 207-242.

59. Robards K., Kerr A.F. & Patsalides E., 1988. Rancidity and its Measurements in Edible Oils and

Snack Foods: A Review. Analyst, 113, 213-224.

60. Ribeiro A.P.B., Corrêa Basso R., Goncalves L.A.G., Gioielli L.A., Oliveira dos Santos A., Pavie

Cardoso L. & Guenter Kieckbusch T., 2012. Physico-chemical properties of Brazilian cocoa

butter and industrial blends. Part II – Microstructure polymorphic behavior and

crystallization characteristics. Grasas y aceites, 63 (1), 89-99.

61. Rodrigues R.C. & Fernandez-Lafuente R., 2010. Lipase from Rhizomucor miehei as a

biocatalyst in fats and oils modification. Journal of Molecular Catalysis B: enzymatic, 66, 15-

32.


62. Rombaut R., De Clercq N., Foubert I. & Dewettinck K., 2009. Triacylglycerol Analysis of Fats

and Oils by Evaporative Light Scattering Detection. Journal of American Oil Chemists’ Society,

86, 19-25.

63. Rossell J.B., 1994. Measurement of rancidity. In: Allen J.C. & Hamilton R.J. (eds.) Rancidity in

foods. Gaithersburg, Aspen Publishers Inc., 22-48.

64. Rozendaal A. & Macrae A.R., 1997. Interesterification of Oils and Fats. In: Gunstone F.D. &

Padley F.B. (eds.) Lipid Technologies and application. Marcel Dekker, New York, 223-263.

65. Salas J.J., Bootello M.A., Martinez-Force E. & Carcés R., 2011. Production of stearate-rich

butters by solvent fractionation of high stearic-high oleic sunflower oil. Food Chemistry, 124,

450-458.

66. Sellappan S. & Akoh C.C., 2000. Enzymatic Acidolysis of Tristearin with Lauric and Oleic Acids

to Prduce Coating Lipids. Journal of American Oil Chemists’ Society, 77 (11), 619-623.

67. Shekarchizadeh H., Kadivar M., Ghaziaskar H.S. & Rezayat M., 2009. Optimization of

enzymatic synthesis of cocoa butter analog from camel hump fat in supercritical carbon

dioxide by response surface method (RSM). The Journal of Supercritical Fluids, 49, 1127-1134.

68. Shieh C., Akoh C.C. & Koehler P.E., 1995. Four-factor Response Surface Optimization of the

Enzymatic Modification of Triolein to Structured Lipids. Journal of American Oil Chemists’

Society, 72 (6), 619-623.

69. Shuang D., Jiang-ke Y. & Yun-jun Y., 2009. Optimization of lipase-catalyzed acidolysis of

soybean oil to produce structured lipids. Journal of Food Biochemistry, 33, 442-452.

70. Smith K.W., 2001. Cocoa butter and cocoa butter equivalents. In: Gunstone F.D. (ed.)

Structured and modified lipids. Bedfordshire, Unilever Research Colworth, 401-422.

71. Sridhar R. & Lakshminarayana G., 1991. Triacylglycerol compositions of some vegetable fats

with potential for preparation of cocoa butter equivalents by high performance liquid

chromatography. Journal of the Oil Technology Association of India, 23, 42-43.

72. Sridhar R., Lakshminarayana G. & Kaimal T.N.B., 1991. Modification of selected Indian

vegetable fats into cocoa butter substitutes by lipase-catalyzed ester interchange. Journal of

American Oil Chemists’ Society, 68 (10), 726-730.

73. Stewart I.M. & Timms R.E., 2002. Fats for chocolate and sugar confectionery. In: Rajak K.K.

(ed.) Fats in food Technology. CRC Press, 159-189.


74. Storgaard S., 2000. New vegetable fats. Sweetec, 4, 5p.

75. Talbot G., 2009b. Vegetable fats. In: Beckett S.T. (ed.) Industrial chocolate manufacture and

use. West Sussex, Wiley-Blackwell, 242-257.

76. Tchobo F.P., Piombo G., Pina M., Soumanou M.M., Villeneuve P. & Sohounhloue D.C.K., 2009.

Enzymatic synthesis of cocoa butter equivalent through transesterification of Pentadesma

butyracea butter. Journal of Food Lipids, 16, 605–617.

77. Timms R.E., 2003. Confectionery fats handbook: properties, production and application.

Bridgwater, The Oily Press, 441 p.

78. Timms R.E., 2005. Fractional crystallization - the fat modification process for the 21st century.

European Journal of Lipid Science and Technology, 107, 48-57.

79. Torbica A., Jovanovic O. & Pajin B., 2006. The advantages of solid fat content determination

in cocoa butter and cocoa butter equivalents by the Karlshamns method. European Food

Research and Technology, 222, 385-391.

80. Toro-Vazquez J.F., Rangel-Vargas E., Dibildox-Alvarado E. & Charo-Alonso M.A., 2005.

Crystallization of cocoa butter with and without polar lipids evaluated by rheometry,

calorimetry and polarized light microscopy. European Journal of Lipid Science and

Technology, 107, 641-655.

81. Tovar L.P., Pinto G.M.F., Wolf-Maciel M.R., Batistella C.B. & Maciel-Filho R., 2011. Short-Path-

Distillation Process of Lemongrass Essential Oil: Physicochemical Characterization and

Assessment Quality of the Distillate and the Residue Products. Industrial & Engineering

Chemistry Research, 50, 8185-8194.

82. Undurrage D., Markovits A. & Erazo S., 2001. Cocoa butter equivalents through enzymatic

interesterification of palm oil midfraction. Process Biochemistry, 36, 933-939.

83. Van Malssen K., Van Langevelde K., Peschar R. & Schenk H., 1999. Phase behaviour and

extended phase scheme of static cocoa butter investigated with real-time X-ray powder

diffraction. Journal of the American Oil Chemists’ Society, 76 (6), 669-676.

84. Vereecken J., Foubert I., Smith K.W. & Dewettinck K., 2009. Effect of SatSatSat and SatOSat

on crystallization of model fat blends. European Journal of Lipid Science and Technology, 111,

243-258.


85. Vereecken J., Foubert I., Smith K.W., Sassano G.J. & Dewettinck K., 2010. Crystallization of

model fat blends containing symmetric and asymmetric monounsaturated triacylglycerols.

European Journal of Lipid Science and Technology, 112, 233-245.

86. Wang H.X., Wu H., Ho C.T. & Weng X.C., 2006. Cocoa butter equivalent from enzymatic

interesterification of tea seed oil and fatty acid methyl esters. Food Chemistry, 97, 661-665.

87. Wassell P., Bonwick G., Smith C.J., Almiron-Roig E. & Young N.W.G., 2010. Towards a

multidisciplinary approach to structuring in reduced saturated fat-based systems – a review.

International Journal of Food Science and Technology, 45, 642-655.

88. Wassell P. & Young N.W.G., 2007. Food applications of trans fatty acid substitutes.

International Journal of Food Science and Technology, 42, 503-517.

89. Widlak N., 1999. Physical properties of fats, oils, emulsifiers. The American Oil Chemists’

Society. 260 p.

90. Xu X., 2003. Engineering of enzymatic reactions and reactors for lipid modification and

synthesis. European Journal of Lipid Science and Technology, 105, 289-304.

91. Xu X., Guo Z., Zhang H., Vikbjerg A.F. & Damstrup M.L., 2006. In: Gunstone F.D. (ed.)

Modifying Lipids for use in food. CRC Press, 234-266.

92. Xu X., Jacobsen C., Skall Nielsen N., Timm Heinrich M. & Zhou D., 2002. Purification and

deodorization of structured lipids by short path distillation. European Journal of Lipid Science

and Technology, 104, 745-755.

93. Xu X., 2000. Production of specific-structured triacylglycerols by lipase-catalyzed reactions: a

review. European Journal of Lipid Science and Technology, 287-303.

94. Xu X., Skands A.R.H., Adler-Nissen J. & Hoy C., 1998. Production of specific structured lipids

by enzymatic interesterification: optimization of the reaction by response surface design.

Fett/Lipid, 100 (10), 463-471.


Appendix I: RSM results Table 19: The level of the factors, and the amount of % SUS and % SUU formed.

Run Substrate

ratio Enzyme

load Water

content Temperature Time Measured

SUS Predicted Measured Predicted

SUS SUU SUU

1 8.00 12.00 3.00 70.00 4.00 66.21 52.31 30.09 34.24

2 7.00 10.00 -0.38 67.50 6.00 63.36 51.79 32.44 38.10

3 8.00 12.00 3.00 70.00 8.00 67.76 58.82 27.06 29.95

4 6.00 8.00 1.00 70.00 8.00 40.69 45.60 50.49 50.21

5 9.38 10.00 2.00 67.50 6.00 63.67 60.34 32.56 30.09

6 6.00 8.00 3.00 70.00 8.00 49.70 46.54 43.48 43.32

7 6.00 8.00 1.00 65.00 4.00 54.72 46.98 39.69 35.85

8 7.00 10.00 2.00 67.50 6.00 64.23 52.90 31.42 35.82

9 6.00 12.00 1.00 65.00 8.00 62.97 59.52 31.42 34.54

10 8.00 8.00 3.00 70.00 4.00 48.33 46.28 44.29 42.79

11 8.00 12.00 1.00 70.00 8.00 58.70 57.89 35.82 36.84

12 8.00 8.00 3.00 70.00 8.00 58.31 52.79 36.66 38.50

13 8.00 12.00 3.00 65.00 8.00 59.98 66.71 34.62 32.79

14 6.00 8.00 1.00 70.00 4.00 36.09 39.09 52.68 54.50

15 6.00 12.00 3.00 65.00 4.00 53.50 53.94 40.41 41.90

16 6.00 12.00 1.00 65.00 4.00 63.15 53.01 32.79 38.83

17 6.00 8.00 3.00 70.00 4.00 46.88 40.03 45.64 47.61

18 6.00 12.00 3.00 70.00 8.00 56.17 52.56 38.12 34.77

19 7.00 10.00 2.00 67.50 6.00 65.14 52.90 30.37 35.82

20 7.00 14.76 2.00 67.50 6.00 59.38 60.07 35.76 37.75

21 6.00 12.00 1.00 70.00 8.00 42.80 51.63 48.40 41.66

22 7.00 10.00 2.00 73.45 6.00 39.81 43.52 49.84 53.84

23 7.00 10.00 2.00 67.50 1.24 44.51 45.16 44.42 40.92

24 4.62 10.00 2.00 67.50 6.00 52.42 45.46 40.06 41.55

25 8.00 8.00 1.00 70.00 8.00 42.91 51.86 49.12 45.40

26 7.00 10.00 4.38 67.50 6.00 54.41 54.01 40.22 33.55

27 8.00 8.00 1.00 65.00 8.00 68.77 59.75 26.44 26.74

28 8.00 8.00 3.00 65.00 8.00 69.59 60.68 25.63 29.81

29 8.00 12.00 1.00 70.00 4.00 56.79 51.38 38.60 41.13

30 7.00 5.24 2.00 67.50 6.00 44.62 45.73 45.99 44.38

31 8.00 8.00 3.00 65.00 4.00 66.46 54.17 30.26 34.10

32 6.00 8.00 1.00 65.00 8.00 62.28 53.49 32.58 31.56

33 7.00 10.00 2.00 67.50 10.76 64.91 60.64 29.57 30.72

34 7.00 10.00 2.00 67.50 6.00 52.96 52.90 40.94 35.82

35 6.00 12.00 3.00 65.00 8.00 53.73 60.45 39.37 37.61

36 6.00 8.00 3.00 65.00 4.00 58.95 47.91 36.22 38.92

37 6.00 8.00 3.00 65.00 8.00 61.57 54.42 32.71 34.63

38 8.00 8.00 1.00 70.00 4.00 38.18 45.35 52.59 49.69

39 8.00 12.00 1.00 65.00 8.00 62.42 65.77 32.75 29.73

40 8.00 12.00 1.00 65.00 4.00 58.59 59.26 37.00 34.01

41 6.00 12.00 3.00 70.00 4.00 51.50 46.05 42.63 39.06

42 7.00 10.00 2.00 67.50 6.00 53.71 52.90 40.70 35.82

43 8.00 12.00 3.00 65.00 4.00 58.72 60.20 36.29 37.08

44 6.00 12.00 1.00 70.00 4.00 40.89 45.12 50.09 45.95

45 8.00 8.00 1.00 65.00 4.00 62.61 53.24 32.98 31.03

46 7.00 10.00 2.00 67.50 6.00 65.72 52.90 29.96 35.82

47 7.00 10.00 2.00 61.55 6.00 61.58 62.28 38.66 35.04


Appendix II: Isothermal crystallization after 1 min

Figure 45: Isothermal crystallization at 20°C after 1 min as visualized by PLM: 0 (a), 20 (b), 40 (c), 60 (d), 80 (e)

and 100% (f) CBE.

a

f e

d c

b


Appendix III: Isothermal crystallization after 1 week, 3 and 5

weeks

Figure 46: Isothermal crystallization at 20°C as visualized by PLM for CB, 20, 40, 60, 80 and 100% CBE. The

microstructure is given after 1 week, 3 and 5 weeks.

Documents

Production of Cocoa Butter Equivalent through Enzymatic