Properties and applications of enzymatically modified cocoa butter. Nathalie De Clercq & Koen Dewettinck UGent Cacaolab
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1 Properties and applications of enzymatically modified cocoa butter Nathalie De Clercq & Koen Dewettinck UGent Cacaolab
2 Modification of CB Cocoa Butter? Cocoa butter DAG Functional ingredient in chocolate?
3 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
4 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
5 Enzymatic modification CB Cocoa Butter Enzymatic glycerolysis Optimization Separation Optimization Cocoa butter DAG Functional ingredient in chocolate? Evaluation
6 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: Water content: 0 to 6 wt% of glycerol mass Response: % DAG Fractional Face-Centered Composite Design: 36 experiments
7 Response surface methodology Regression calculations: Quadratic model Regression coeffiencts Factor water removed no influence Variable Regression coefficients p-values Intercept A: Reaction time B: Enzyme load 5.03 < C: Temperature 4.86 < D: Substrate molar ratio AC AD BC A² D²
8 C : T e m p e r a t u r e Response surface methodology % DAG B: Enzyme Load
9 D : S u b s t r a t e m o la r r a t io Response surface methodology % DAG A: Reaction time
10 C : T e m p e r a t u r e Response surface methodology % DAG A: Reaction time
11 C : T e m p e r a t u r e Response surface methodology Design expert: optimal conditions: 53% DAG Reaction time: hours Enzyme load: 15% Temperature: 75 C Design-Expert Software Factor Coding: Actual Substrate molar ratio: 1.35 % DAG % DAG Prediction X1 = A: Reaction time X2 = C: Temperature Actual Factors B: Enzyme Load = D: Substrate molar ratio = 1.35 E: Water content = A: Reaction time
12 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
13 Effect of reaction time (a) 55 C (b) 75 C % % TAG DAG MAG h 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 h 5h 6h 7h 8h 9h Time (hours)
14 Effect of substrate ratio (b) (a) DAG MAG % (MAG + DAG) % 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
15 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
16 Cocoa butter DAG Cocoa Butter Enzymatic glycerolysis Separation: short path distillation Cocoa butter DAG
17 Heat Flow (W/g) 0.0 Cocoa butter DAG Sample: bis non iso B Size: mg Method: non iso 5-5 DSC File: C:...\non iso\0-100 Bis non iso b Operator: Liesbeth Run Date: 09-May :46 Instrument: DSC Q1000 V9.9 Build Low melting High melting C C Exo Up Temperature ( C) Universal V4.7A TA Instruments
18 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
19 Heat Flow (W/g) Chocolates containing DAG % 1,25 % 2,5 % 5 % 7,5 % ,5 % 25 % Exo Up Temperature ( C) Universal V4.7A TA Instruments
20 Chocolate flow behaviour Flow behaviour: plate-plate geometry at 40 C Shear stress (Pa) % 1,25% 2,5% 5% 7,5% 10% 12,5 % 25% Shear rate (1/s)
21 Chocolate flow behaviour Casson Yield(a) stress Casson Viscosity (b) 50 1, ,15 Casson Yield Stess (Pa) Casson Viscosity (Pa.s) 1,10 1,05 1,00 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
22 Texture analysis Chocolate were hand tempered and compared with non tempered Texture analysis after 24 hours
23 Texture analysis Three point bend test (a) Penetration test (b) Non tempered Tempered Maximum Load (N) Hardness (N) % DAG % DAG
24 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
25 Acknowledgements: Barry-Callebaut FWO Flanders
26 Properties and applications of enzymatically modified cocoa butter Nathalie De Clercq & Koen Dewettinck UGent Cacaolab
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