Properties and applications of enzymatically modified ...

Properties and applications of enzymatically modified cocoa butter

Nathalie De Clercq & Koen DewettinckUGent Cacaolab

Modification of CB

Cocoa Butter

Cocoa butter DAG

Functional ingredient in chocolate ?

?

Various routes for the production of DAG

• Glycerolysis between TAG and glycerol, chemical or enzymatically (with or without organic solvent)

• Esterification of fatty acids to glycerol (with or without organic solvents)

• Selective hydrolysis of TAG

• Combination of the above described methods

Modification of CB

LIMITATIONS• Simple reaction• Substrate cocoa butter• Food applications• Easy to control

Reaction toolbox

• Glycerolysis Chemical Enzymatic

• Esterification Chemical Enzymatic

• Hydrolysis

Enzymatic glycerolysis

Modification of CB

Enzymatic modification CB

Optimization

Separation

Cocoa Butter


Optimization

Cocoa butter DAG

Functional ingredient in chocolate ? Evaluation

Enzyme

• Candida antarctica= Novozym 435

Response surface methodology

• Variables

Reaction time: 3 to 15 hours

Reaction temperature: 40°C to 75°C

Enzyme load: 3 to 15 wt% of oil mass

Substrate molar ratio, oil/glycerol: 0.25-2.00

Water content: 0 to 6 wt% of glycerol mass

• Response: % DAG

• Fractional Face-Centered Composite Design: 36 experiments

Experimental set up

Regression calculations: Quadratic model

Regression coeffiencts

• Factor water removed no influence


Variable Regression coefficients p-values

Intercept 45.54 0.001

A: Reaction time 2.88 0.0033

B: Enzyme load 5.03 <0.0001

C: Temperature 4.86 <0.0001

D: Substrate molar ratio 1.89 0.0436

AC -1.79 0.0694

AD 2.97 0.0041

BC -2.73 0.0076

A² -4.39 0.0404

D² -4.83 0.0255

Design-Expert® SoftwareFactor Coding: Actual% DAG

Design Points51.73

16.02

X1 = B: Enzyme LoadX2 = C: Temperature

Actual FactorsA: Reaction time = 9.00D: Substrate molar ratio = 1.13E: Water content = 3.00

3.00 6.00 9.00 12.00 15.00

45.00

51.00

57.00

63.00

69.00

75.00% DAG

B: Enzyme Load

C:

Te

mp

era

ture

34

36

38

40

42

44

46

48

50

10



51.73

16.02

X1 = A: Reaction timeX2 = D: Substrate molar ratio

Actual FactorsB: Enzyme Load = 9.00C: Temperature = 59.59E: Water content = 3.00

3.00 6.00 9.00 12.00 15.00

0.25

0.69

1.13

1.56

2.00% DAG

A: Reaction time

D:

Su

bs

tra

te m

ola

r ra

tio

34

36 36

36

38

38

38

40

42

44

46



Design Points51.73

16.02

X1 = A: Reaction timeX2 = C: Temperature

Actual FactorsB: Enzyme Load = 9.00D: Substrate molar ratio = 1.13E: Water content = 3.00

3.00 6.00 9.00 12.00 15.00

45.00

51.00

57.00

63.00

69.00

75.00% DAG

A: Reaction time

C:

Te

mp

era

ture

34

36

38

40

42

44

46

48

10



Design expert: optimal conditions: 53% DAG

• Reaction time: 10.26 hours

• Enzyme load: 15%

• Temperature: 75°C

• Substrate molar ratio: 1.35Design-Expert® SoftwareFactor Coding: Actual% DAG

51.73

16.02

X1 = A: Reaction timeX2 = C: Temperature

Actual FactorsB: Enzyme Load = 15.00D: Substrate molar ratio = 1.35E: Water content = 3.00

3.00 6.00 9.00 12.00 15.00

45.00

51.00

57.00

63.00

69.00

75.00% DAG

A: Reaction time

C:

Te

mp

er

atu

re

40

42

44

4648 50

50

52

Prediction 53.058

Reaction optimization

Further optimization and model verification

Temperature

55 C

75 C

80 C

Substrate molar ratio

0,7 – 1,12 - 2

Reaction Follow up: 4 h – 5 h – 6 h – 7 h – 8 h – 9 h

(a) 55°C

Time (hours)

4h 5h 6h 7h 8h 9h

%

0

10

20

30

40

50

TAG

DAG

MAG

(b) 75°C

Time (hours)

4h 5h 6h 7h 8h 9h

%

0

10

20

30

40

50

(c) 80°C

Time (hours)

4h 5h 6h 7h 8h 9h

%

0

10

20

30

40

50 TAG

DAG

MAG

Effect of reaction time

• 55°C: equilibrium after 7 hours• 75°C: equilbrium after 4 hours RSM

Effect of substrate ratio

More glycerol (lower substrate ratio): higher conversion degree, higheramount of MAG but lower amount of DAG

(a)

55

°C; S

R 0

,7

55

°C; S

R 1

,12

55

°C; S

R 1

,5

75

°C; S

R 0

,7

75

°C; S

R 1

,12

75

°C; S

R 1

,5

80

°C; S

R 0

,7

80

°C; S

R 1

,12

80

°C; S

R 1

,5

%

0

5

10

15

20

25

30

35

40

45

50

55DAG

MAG

(b)

55

°C; S

R 0

,7

55

°C; S

R 1

,12

55

°C; S

R 1

,5

75

°C; S

R 0

,7

75

°C; S

R 1

,12

75

°C; S

R 1

,5

80

°C; S

R 0

,7

80

°C; S

R 1

,12

80

°C; S

R 1

,5

% (

MA

G +

DA

G)

0

10

20

30

40

50

60

70

80

90

No water necessary

Reaction time: 6 hours: a reasonable time to obtain equilibrium

Reaction temperature :70°C being still in the range of maximum enzyme activity

Enzyme load: 15 wt% of the oil mass: high amount of enzyme didn’t had negative effects

Substrate molar ratio: 1.12 combination of a reasonable conversion with a reasonable amount of glycerol

Reaction optimization

Cocoa butter DAG

Separation: short path distillation

Cocoa Butter


Cocoa butter DAG

33.12°C

60.53°C

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

He

at

Flo

w (

W/g

)

-20 0 20 40 60 80

Temperature (°C)

Sample: 0-100 bis non iso BSize: 8.2400 mgMethod: non iso 5-5

DSCFile: C:...\non iso\0-100 Bis non iso bOperator: LiesbethRun Date: 09-May-2011 10:46Instrument: DSC Q1000 V9.9 Build 303

Exo Up Universal V4.7A TA Instruments

Cocoa butter DAG

“Low melting” “High melting”

Chocolates

• 0, 1.25, 2.5, 5, 10, 12.5 and 25 % DAG on fat base

• 0.4% to 8.75% DAG on product base

Melting behaviour

Rheological behaviour

Texture analysis

Chocolates containing DAG

Chocolates containing DAG

-1.2

-0.7

-0.2

0.3

He

at

Flo

w (

W/g

)

20 30 40 50 60

Temperature (°C)–––––––– – – –––––– ·––– – –––– –––––––– ––– –– –Exo Up Universal V4.7A TA Instruments

0 %

1,25 %

2,5 %

5 %

7,5 %

12,5 %

25 %

Shear rate (1/s)

0 20 40 60 80 100

She

ar

str

ess (

Pa)

0

100

200

300

400

500

0%

1,25%

2,5%

5%

7,5%

10%

12,5 %

25%

Chocolate flow behaviour

Flow behaviour: plate-plate geometry at 40°C

(a)

% CB DAG

0,0 2,5 5,0 7,5 10,0 12,5

Casson Y

ield

Ste

ss (

Pa)

10

15

20

25

30

35

40

45

50

(b)

% CB DAG

0,0 2,5 5,0 7,5 10,0 12,5

Cass

on V

isco

sity

(P

a.s

)

0,85

0,90

0,95

1,00

1,05

1,10

1,15

1,20

Chocolate flow behaviourCasson Yield stress Casson Viscosity

• Up to 5% no influence on rheological parameters• > 7.5%: DAG interact at the interphasemicelle formation and/or

multilayers increased yield stress• > 7.5%: higher viscosity: remaining crystals at 40°C

Chocolate were hand tempered and compared with non tempered

Texture analysis after 24 hours

Texture analysis

(a)

% DAG

0 5 10 15 20 25

Maxim

um

Load (

N)

0

20

40

60

80

100

Non tempered

Tempered

(b)

% DAG

0 5 10 15 20 25 30

Hard

ness (

N)

0

50

100

150

200

250

Texture analysis

Three point bend test Penetration test

Enzymatic glycerolysis is a good technique to produce CB based DAG

CB + enzyme + glycerol 50% yield DAG

CB DAG in chocolate

• Up to 5%: limited influence on chocolate properties

• Possibility to adjust yield stress with a CB based emulsifier

Conclusions

Acknowledgements:

Barry-Callebaut

FWO Flanders

Properties and applications of enzymatically modified cocoa butter

Nathalie De Clercq & Koen DewettinckUGent Cacaolab

Properties and applications of enzymatically modified ...

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