Properties and applications of enzymatically modified cocoa butter Nathalie De Clercq & Koen Dewettinck UGent Cacaolab
Modification of CB Cocoa Butter? Cocoa butter DAG Functional ingredient in chocolate?
Modification of CB 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 Reaction toolbox Glycerolysis Chemical Enzymatic Esterification Chemical Enzymatic Hydrolysis LIMITATIONS Simple reaction Substrate cocoa butter Food applications Easy to control Enzymatic glycerolysis
Enzymatic modification CB Cocoa Butter Enzymatic glycerolysis Optimization Separation Optimization Cocoa butter DAG Functional ingredient in chocolate? Evaluation
Experimental set up 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
Response surface methodology 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
C : T e m p e r a t u r e Response surface methodology 75.00 % DAG 69.00 50 48 63.00 57.00 42 44 10 46 51.00 45.00 40 38 36 34 3.00 6.00 9.00 12.00 15.00 B: Enzyme Load
D : S u b s t r a t e m o la r r a t io Response surface methodology 2.00 34 36 % DAG 38 1.56 1.13 44 46 42 0.69 40 38 36 38 36 0.25 3.00 6.00 9.00 12.00 15.00 A: Reaction time
C : T e m p e r a t u r e Response surface methodology 75.00 % DAG 69.00 48.13 63.00 10 46 57.00 51.00 36 38 40 42 44 34 45.00 3.00 6.00 9.00 12.00 15.00 A: Reaction time
C : T e m p e r a t u r e Response surface methodology Design expert: optimal conditions: 53% DAG Reaction time: 10.26 hours Enzyme load: 15% Temperature: 75 C Design-Expert Software Factor Coding: Actual Substrate molar ratio: 1.35 % DAG 51.73 16.02 75.00 69.00 % DAG Prediction 53.058 52 X1 = A: Reaction time X2 = C: Temperature Actual Factors B: Enzyme Load = 15.00 D: Substrate molar ratio = 1.35 E: Water content = 3.00 63.00 57.00 51.00 42 44 46 48 50 50 40 45.00 3.00 6.00 9.00 12.00 15.00 A: Reaction time
Reaction optimization Further optimization and model verification Reaction Follow up: 4 h 5 h 6 h 7 h 8 h 9 h Temperature 55 C 75 C 80 C Substrate molar ratio 0,7 1,12-2
Effect of reaction time (a) 55 C (b) 75 C 50 50 40 40 % 30 20 % 30 20 TAG DAG MAG 10 10 0 4h 5h 6h 7h 8h 9h 0 4h 5h 6h 7h 8h 9h Time (hours) Time (hours) (c) 80 C 55 C: equilibrium after 7 hours 75 C: equilbrium after 4 hours RSM % 50 TAG DAG MAG 40 30 20 10 0 4h 5h 6h 7h 8h 9h Time (hours)
Effect of substrate ratio (b) (a) 90 55 DAG MAG 80 50 % (MAG + DAG) 70 60 50 40 30 20 10 % 45 40 35 30 25 20 15 10 5 0 0 80 C; SR 1,5 80 C; SR 1,12 80 C; SR 0,7 75 C; SR 1,5 75 C; SR 1,12 75 C; SR 0,7 55 C; SR 1,5 55 C; SR 1,12 55 C; SR 0,7 80 C; SR 1,5 80 C; SR 1,12 80 C; SR 0,7 75 C; SR 1,5 75 C; SR 1,12 75 C; SR 0,7 55 C; SR 1,5 55 C; SR 1,12 55 C; SR 0,7 More glycerol (lower substrate ratio): higher conversion degree, higher amount of MAG but lower amount of DAG
Reaction optimization 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
Cocoa butter DAG Cocoa Butter Enzymatic glycerolysis Separation: short path distillation Cocoa butter DAG
Heat Flow (W/g) 0.0 Cocoa butter DAG Sample: 0-100 bis non iso B Size: 8.2400 mg Method: non iso 5-5 DSC File: C:...\non iso\0-100 Bis non iso b Operator: Liesbeth Run Date: 09-May-2011 10:46 Instrument: DSC Q1000 V9.9 Build 303-0.2-0.4-0.6 Low melting High melting -0.8 33.12 C 60.53 C -1.0-20 0 20 40 60 80 Exo Up Temperature ( C) Universal V4.7A TA Instruments
Chocolates containing DAG 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
Heat Flow (W/g) Chocolates containing DAG 0.3 0 % 1,25 % 2,5 % 5 % 7,5 % -0.2 12,5 % 25 % -0.7-1.2 20 30 40 50 60 Exo Up Temperature ( C) Universal V4.7A TA Instruments
Chocolate flow behaviour Flow behaviour: plate-plate geometry at 40 C Shear stress (Pa) 500 400 300 200 0% 1,25% 2,5% 5% 7,5% 10% 12,5 % 25% 100 0 0 20 40 60 80 100 Shear rate (1/s)
Chocolate flow behaviour Casson Yield(a) stress Casson Viscosity (b) 50 1,20 45 1,15 Casson Yield Stess (Pa) 40 35 30 25 20 Casson Viscosity (Pa.s) 1,10 1,05 1,00 0,95 15 0,90 10 0,85 0,0 2,5 5,0 7,5 10,0 12,5 0,0 2,5 5,0 7,5 10,0 12,5 % CB DAG Up to 5% no influence on rheological parameters % CB DAG > 7.5%: DAG interact at the interphase micelle formation and/or multilayers increased yield stress > 7.5%: higher viscosity: remaining crystals at 40 C
Texture analysis Chocolate were hand tempered and compared with non tempered Texture analysis after 24 hours
Texture analysis Three point bend test (a) Penetration test (b) 100 80 Non tempered Tempered 250 200 Maximum Load (N) 60 40 Hardness (N) 150 100 20 50 0 0 5 10 15 20 25 0 0 5 10 15 20 25 30 % DAG % DAG
Conclusions 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
Acknowledgements: Barry-Callebaut FWO Flanders
Properties and applications of enzymatically modified cocoa butter Nathalie De Clercq & Koen Dewettinck UGent Cacaolab