properties and applications of enzymatically modified
TRANSCRIPT
Properties and applications of enzymatically modified cocoa butter
Nathalie De Clercq & Koen DewettinckUGent Cacaolab
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
Enzymatic glycerolysis
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
Response surface methodology
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
Response surface methodology
Design-Expert® SoftwareFactor Coding: Actual% DAG
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
Response surface methodology
Design-Expert® SoftwareFactor Coding: Actual% DAG
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
Response surface methodology
Response surface methodology
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
Enzymatic glycerolysis
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