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  • Cocoa butter equivalents (CBE) are derived from reconstructing of triacylglycerols (TAGs) within oils.
  • As the demand for cocoa butter (CB) increases, the global supply decreases due to climatic conditions. 
  • In addition, in order to supply cocoa butter, there have been inhumane practices of child labor, therefore, resorting to synthesizing an equivalent assists in preventing such inhumanities.
  • The main TAGs within olive oil and palm oil that will be prioritized during characterization:
    • 1-palmitoy-2,3-dioleoyl glycerol (POO)
    • 1,3-dipalmitoyl-2-oleyl-glycerol (POP)
    • (3)-palmitoyl-3(1)-stearoyl-2-oleoyl-glycerol (POS)
    • 1,3-distearoyl-2-oleoyl-glycerol (SOS)
    • 1-stearoyl-2,3-dioleoyl-glycerol (SOO)
    • 1-palmitoy-2,3-dioleoyl glycerol is the target TAG in terms of our kinetic study due to it having the highest percent conversion.
  • Chocolates used for Quantitative study: milk, white, and dark (90% cocoa).

Enzymatic Synthesis of Cocoa Butter Equivalent (CBE) using Oils and the Quantification of Fatty Acid Methyl Esters (FAMEs) in Chocolate Products

 Rose Tomson, Jacqueline Medina, Hala Muhiar, Rahilah Alkak, A. Paige Wicker, Vo Quang Huy Phan, Frank W. Foss Jr., Kevin A. Schug

 Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX

Column

FAMEWAX: 30 m, 0.25 mmID x 0.25 µm (Restek)

Oven Temp.

200 °C (hold for 1 min.) to 250 °C at 30°C/min (hold for 4 min.)

Injector Temp.

250 °C

Injection Volume

1.0 µL

Carrier Gas

Helium

Split Ratio

20:1

Ion Source Temp. and Interface Temp.

Ion Source: 150 °C, Interface: 230 °C

 Results and Expected Outcomes

Acknowledgements

Methods

Procedures

Future Works

Synthesis of CBE

Quantitative Methyl Esterification of Chocolate Fats

Gas Chromatography Mass Spectrometry (GC-MS) Method for Quantitative Study

References

(1) Mohamed, I.O. Appl Biochem Biotechnol. 2015, 175, 757–769. 

(2) Amorim, T. L. et al. J. Agric. Food Chem. 2019, 67 (40). 

​

  • Continue kinetic study with the 1:3 olive oil to palmitic-stearic fatty acid mixture.
  • Run 1:3 OO:PSFAM under GC-MS analysis once method is determined.
  • Regarding the quantitative study, it is possible to attempt to go further and separate the fatty acids into cis and trans fatty acids before methylation.

Objectives

  • Quantify fatty acid methyl esters in chocolates
  • Synthesize Cocoa Butter equivalents (CBE)
  • Compare % peak areas TAGs

Quantitative

FAMEs

C13

C15

Palmitic

C13

Stearic

C5

Introduction

Sn,1-3 Lipase

KOH

Palmitic acid

Stearic acid

Oleic acid

CBE

+

Melting Point (MP) Apparatus

Quantitative Study: Quantification of Fatty Acid Methyl Esters in Chocolate

C15

=C9

POS

Synthesis: Enzymatic Synthesis of a Cocoa Butter Equivalent

Kinetics

Table 1. Concentrations of the three target FAMEs in chocolate in ppm.

OO to PSFAM Ratios

Starting point

End point

1:1

46°C

48°C

1:2

49°C

60°C

1:3

34°C

49°C

1:4

54°C

58°C

1:5

54°C

59°C

60°C

160RPM

0°C

Schematics

PO to PSFAM Ratios

Starting point

End point

1:1

46°C

49°C

1:2

48°C

53°C

1:3

50°C

53°C

1:4

51°C

55°C

Time Intervals

5 – 320 Minutes.

Doubling the previous time as the next time point.

1:3 OO:PSFAM

Differential Scanning Calorimetry (DSC) Method

  • Anna Romero
  • Joisha Girme
  • Ravi Ramkissoon
  • Dr. Kayunta Johnson-Winters
  • Nazmee Parveen

Synthesis

Potential GC-MS Method for Characterization of Synthesized CBE Samples

Column

Rxi-PAH: 60 m, 0.25 mmID x 0.1 µm (Restek)

Oven Temp.

Isothermal: 280 °C (hold 35 min.)

Injector Temp.

250 °C

Injection Volume

1.0 µL

Carrier Gas

Helium

Split Ratio

10:1

Ion Source Temp. and Interface Temp.

Ion Source: 250 °C, Interface: 250 °C

Start at -40°C

Rate 10°C/min to 80°C, Hold for 15 minutes

Bring down to room Temperature

 

Concentration of Methyl palmitate (ppm)

Concentration of Methyl stearate (ppm)

Concentration of Methyl oleate (ppm)

Milk

9.98

10.2

10.2

White

13.4

15.0

14.8

Dark

13.0

16.8

15.7

Palmitate

Oleate

Stearate

Figure 3. GC chromatogram displaying dark chocolate sample.

Figure 1. Endothermic peak for DSC1

Figure 5. Kinetic study from the literature1

Figure 2. Calibration curve from FAMEs standard

Figure 4. DSC determined that the Endothermic peak of natural CB is at 37.81°C

Expected Kinetic Results

1

2

3

1

Methyl palmitate

2

Methyl stearate

3

Methyl oleate

(3) Buchgraber, M. et al. J. Agric. Food Chem. 2004, 52, 3855−3860.

Gives CB unique melting characteristics

POO has the largest change overtime

CB MP

~37°C

Table 2. MP ranges of both Olive oil and Palm oil at varying concentration

DSC Thermogram for Natural Cocoa Butter (CB)

H

H