Production of Cocoa Butter Equivalent through Enzymatic Acidolysis · 2012. 12. 5. · Production of Cocoa Butter Equivalent through Enzymatic Acidolysis II Woord vooraf Deze masterproef

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.

Production of Cocoa Butter Equivalent through Enzymat

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