International Food Research Journal 23(1): 47-54 (2016)
Journal homepage: http://www.ifrj.upm.edu.my

Effect of extraction methods on yield, oxidative value, phytosterols andantioxidant content of cocoa butter
Roiaini, M., 1Seyed H. M., 2Jinap, S. and 1*Norhayati, H.

1

Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia
2
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia

1

Article history

Abstract

Received: 19 May 2015
Received in revised form:
23 June 2015
Accepted: 7 July 2015

Cocoa beans are rich in numbers of beneficial bioactive compounds such as phenolics andphytosterols, which benefits to human being. The suitable extraction method is needed to
produce high quality and quantity of cocoa butter and other bioactive compounds. There aremany extraction method to extract these compounds such as Soxhlet extraction, supercriticalfluid extraction, ultrasound extraction method and others. The objective of this study is todetermine the effectiveness of the different extraction methods producing high yields of cocoa
butter, lower oxidative value, stable phytosterols and antioxidant content. The cocoa beans were
subjected to different extraction methods such as Soxhlet extraction (SE), Ultrasonic extraction
method (USE), Supercritical carbon dioxide (SCO2) and Supercritical carbon dioxide with cosolvent (SCO2-Ethanol). Cocoa butter extracted using SCO2-Ethanol has significantly (p<0.05)
obtained highest cocoa butter yield (37.05%) and phytosterols content (6441 μg/g of extract)compared to SE (28.87% and 4960 μg/g of extract), SCO2 (31.32% and 5492 μg/g of extract)and USE (34.81% and 5106 μg/g of extract). Meanwhile, the oxidative value of SCO2-Ethanol
was significantly (p<0.05) obtained lowest value compared to other extraction methods.
Extraction method are crucial in cocoa industry to minimise the cost during processing, obtainmaximum extraction yield and preserve the bioactive compounds thus will improve the valueof cocoa butter.improve the value of cocoa butter.

Keywords
Soxhlet extraction
Ultrasonic extraction
Supercritical fluid
extraction
Phytosterols
Antioxidant
Cocoa butter© All Rights Reserved

Introduction
Cocoa butter is responsible for the melting
properties of chocolate and was obtained from cocoa
beans around 50–57% yield dry weight (Steinberg
et al., 2003). It contain omega-6 and omega-9 fatty
acids, and also natural antioxidants such as alphatocopherol and phytosterols, as well as nutrients
which support mood and the immune system (Norton
et al., 2009). The major fatty acids in cocoa butter
are palmitic acid (C16) 25–33.7%, stearic acid
(C18:0) 33.7–40.2%, oleic acid (C18:1) 26.3–35%
and linoleic acid (C18:2) 1.7–3% which contributeto about 98% of the total fatty acid (Asep et al.,2008; Bracco, 1994). The uniqueness of cocoa butter
includes brittle at room temperature and fast liquefies
at body temperature. Due to their remarkablefunctional properties, cocoa butter was essentiallybeing used in the food, cosmetic and pharmaceuticalindustries. In the food industry, the mild texture,mouth feel, flavour release and shimmer of chocolate
products were produced from cocoa butter (Liendo etal., 1997; Schilchter-Aronhime and Garti, 1988).
*Corresponding author.
Email: aryatihussain@upm.edu.myTel: +603-89468536

Li and Hartland (1996) stated that hexane has
been broadly used to extract cocoa butter in solvent
extraction similar to other fats and oils from oilcontained sources. The application of organic
solvents has raised the concern for the health andsafety hazards. The cleaner extraction method waschosen due to great responsibility over disposalof such toxic organic solvents and their effect on
the environment (Jahurul et al., 2014). In contrast,
hydraulic method which has been widely used by
the cocoa industries always initiates contaminants
into the cocoa butter that must be removed later. This
places a new alternative in acquiring cocoa butter toemerge clean and efficient technologies.
Latest extraction techniques suitable for cocoa
butter extraction are ultrasound-assisted extraction(USE), pressurized liquid extraction, microwave
extraction and supercritical fluid extraction (Zuoaga
et al., 1998; Schroeter et al., 1999; Hawthorne etal., 2000; Giergielewicz-Mozajska et al., 2001).
The differentiation of traditional methods withsupercritical extraction, it is practical on producing
a good product and has the ability to obtain higher

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Roiaini et al./IFRJ 23(1): 47-54

yields and a superior quality cocoa butter replacers
blends (Zaidul et al., 2006; Zaidul et al., 2007a,c). On the other hand, USE has been recognized to
extract wide range of herbal and other plant extracts.
The propagation of ultrasound pressure waves,develop from cavitation phenomena by ultrasound,resulting in enhancement of extraction (Vilkhu etal., 2008). Ultrasounds cause mechanical effects
due to the agitation of the solvent, the increaseof contact surface area between solid and solvent
and thus, greater infiltration of the solvent into thematrix (Zhang Zh-Sh et al., 2008; Kowalski andWawrzykowski 2009; Shalmash 2009). Recently, it
was shown that the yield of oil by USE was similar to
Soxhlet extraction (Da Porto et al., 2013).
The extraction of oil using different extraction
methods may produce oils with improved
characteristics such as able to produce high yieldand preserve the bioactive compounds. Therefore,
the goal of this research was to investigate theimpact of the different extraction methods of cocoa
bean in obtaining maximum yield of cocoa butter,
phytosterols and antioxidant content and lower
oxidation value,.
Materials and Methods
Materials
The cocoa beans were purchased from Lembaga
Koko Jengka, Pahang, Malaysia. Cocoa shells
were removed manually from cocoa beans and thenibs were smashed approximately below 1 mmwith a mortar and pestle before being analysed todetermine the yield, oxidative value, phytosterols
and antioxidant content. The nibs were stored in adark closed container to prevent from humidity andlight at room temperature (24°C).
Extraction procedures
The cocoa nibs were hydrolyzed with dilutehydrochloric acid (25%) and filtered (Whatman
filter paper, 8.0 µm) before extraction processes. The
cocoa nibs were extracted using applied methods
such as (Soxhlet extraction, Ultrasonic extraction,
Supercritical extraction CO2 and ethanol. The
cocoa butter obtained from the extraction procedure
mentioned below was analysed for comparison ofoxidative value, phytosterols and antioxidant content.
The yield of cocoa butter was calculated using theformula below:
					

x 100

Soxhlet extraction
Approximately 5 g of cocoa nibs was weighted
into the thimble. Then, 150 ml of ethanol (99.5%)
added in 120 ml round bottom flask and refluxedfor 6 h using Soxhlet apparatus. The extracts were
filtered and concentrated under reduced pressure
until dryness (AOCS 1998).
Supercritical extraction: SCO2 and SCO2-Ethanol
Supercritical extraction consist of Intelligent
HPLC Pump Model PU-1580 (Jasco Corporation,
Tokyo, Japan). A Back Pressure Regulator (BPR)
Model BP- 1580-81 (Jasco Corporation, Tokyo,
Japan) was used to control the extraction pressure.
Separation was controlled using Back Pressure
Regulator (BPR) (Model BP-1580-81, Jasco
Corporation, Tokyo, Japan). Approximately 20 g ofcocoa nibs were loaded into 50 ml extraction vessel.
SCO2 was performed using carbon dioxide alone at
60°C and 35 MPa, while SCO2-Ethanol using both
CO2 and ethanol (25%) at 60°C and 35 MPa and flowrate of ethanol was 2 ml/ min (Asep et al., 2008).
Ultrasonic extraction
Ultrasonic extractions were performed with
an ultrasound cleaning bath–Delta/ DC150H.
Approximately 20 g of cocoa nibs were extracted with
200 ml of ethanol (99.5%) in an ultrasonic apparatus.
The temperature of the bath was controlled at 30˚C.
The cocoa nibs were immersed in ethanol andhomogenized a few minutes before ultrasonicatedfor 1 h. After the process were repeated twice, thesolvent was collected by filtering the extract using
Whatman filter No. 1. The solvent containing cocoa
butter then were applied with rotary evaporator to
remove ethanol (Mariod et al., 2011).
Oxidative analysis
Iodine value (IV)The IV measures the number of reactive double
bonds present in cocoa butter. A higher IV number
indicates more double bonds in the cocoa butter. IV
was determined according to AOCS method Cd 1d92 (1993b).
Peroxide value (PV)The PV measures the oxidation processes in oil.
PV of cocoa butter was determined according to
AOCS method Cd 8-53 (1993c).
Acid value (AV)The acid value (which is twice the free fatty acid
(FFA) value) measures the amount of fatty acids

Roiaini et al./IFRJ 23(1): 47-54

separated from glycerol molecules. The amount of
free fatty acid present in cocoa butter was estimatedby determining the quantity of alkali that must be
added to the fat to become neutral. Acid value of
cocoa butter was determined according to AOCS
method Ca 5a-40 (1993).
Determination of phytosterols
The phytosterols composition of the cocoa butter
was determined by Careri et al. (2001). About 200
mg of cocoa butter was added into 50 ml roundbottomed flask followed by the addition of 250 μl of
5-α cholestane (1000 μg/ml) in methanol as internal
standard and 2 ml of 2 M KOH in methanol. The
mixture was heated under reflux at 90˚C for 1 h. After
that, 4 ml of water and ethyl acetate were added to the
mixture. The aqueous phase was washed three times
with diethyl ether. Finally, the diethyl ether solution
was dried with sodium sulfate anhydrous, filteredand then dried with a nitrogen stream. Solid phaseextraction (SPE) was applied to perform the clean-up
procedure. Silica gel (packing 500 mg/6 ml, Silicycle)
SPE tubes were used and the mixture was dissolvedin 5 ml hexane-ethyl acetate (95:5, v/v) and 6 ml ofhexane-ethyl acetate (60:40) v/v) to perform washingstep. The eluate was dried under a nitrogen stream andre-dissolved in 1 ml of hexane. The extracts obtained
were analysed by HPLC with UV detection (HPLCUV). HPLC Waters system consisted of an auto
sampler and a spectrophotometric UV-Vis variablewavelength detector. Separation was carried out using
C18 column (150x2.1 mm, 5 μm) (Silicycle) under
isocratic condition with a mixture of acetonitrilewater (86:14, v/v). The operative wavelength was
set at 200.4 nm. The injection volume was 1 μl. The
samples were injected triplicate.
Determination of antioxidant activity
Approximately 1±0.01 g of cocoa butter wasadded with 10 ml of methanol in water (90:10). Thenthe solution was placed in an ultrasonic bath for 5 min.
Subsequently, the sample was centrifuged for 10 min
at 4,000 rpm at 24°C. The extraction procedure wascarried out three times. All methanol extracts werecombined, filtered and treated by rotary evaporator
to dryness. The extracts were kept at -20°C, and justbefore spectrophotometric determination, they weremixed with 2.5 ml of the concentration of methanol(10:90) and centrifuged as previously. The analysis
was done in triplicate (Malićanin et al., 2014).
DPPH assay
About 3.2 mg of DPPH powder was diluted in
80% methanol. Then, 3.9 ml of DPPH solution was

49

added to 0.1 ml of extracts. The mixture was kept indark for 2 h. The absorbance was measured at 517
nm.
Total phenolic content (TPC)
About 500 μl Folin-Ciocalteu and 1.5 ml
sodium carbonate (20%) were added in 100 μl of
the extracts. The mixture was incubated in the dark
at room temperature for 90 min. The absorbancewas measured at 730 nm. A calibration curve wasobtained against standard Gallic acid. The results
were expressed as mg of Gallic acid per 100 g of oil.
Statistical analysis
All analysis were done in triplicate (n=3) and thedata were statistically analyzed by one-way analysis
of variance (ANOVA) procedure, using Minitab
software. Significant differences (p<0.05) between
means were determined.
Results and Discussion
From the preliminary study, ethanol was used assolvent since the yield of phytosterols and antioxidant
value was higher (4974 mg/100 g of extract, DPPH;
64.87% and TPC; 22.38 mg GAE/100 g of extract)compared to hexane, petroleum ether and 2-propanol.
Yield determination
The SCO2-Ethanol (37.05%) and USE (34.81%)
produced significantly (p<0.05) higher yield of
cocoa butter compared to SE and SCO2 (28.87% and31.32%) (Table 1). The physical and chemical of
cocoa butter is very complex and changes dependingon the processing it receives. The greatest loss of
cocoa butter from cocoa bean was observed in SE
due to high temperature applied and no agitation
provided during extraction. Each extraction wasapplied with different temperature. The higher
temperature was set on SE at 78°C, while both SCO2
and SCO2-Ethanol was set at 60°C and USE was set
at 30°C. Different temperature applied will effectdifferent yield of cocoa butter. The major fatty acidpresent in cocoa butter are oleic acid (34.5%), stearicacid (34.5%) and palmitic acid (26.0%) (Jahurul etal., 2014). Stearic acid have a nonpolar chain that
confers solubility in organic solvent. Thus, cocoa
butter can be extract using solvent but the amount ofyield obtained was influenced by the processing of
the extraction.
Asep et al. (2008) stated that SCO2-Ethanol has
a lot of benefit due to its potential to obtain products
clear from sample residues, fast and alternative
methods for environmental reasons by the minimum

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Roiaini et al./IFRJ 23(1): 47-54

Table 1. Yield percentage, Iodine value, Peroxide value and Acid value in cocoa butter using different
extraction methods

Results were expressed as means ± SD for triplicates measurement. Means in the same column with different
letters were significantly different (ρ < 0.05).

usage of large solvent volumes. The higher yield of
cocoa butter obtained from SCO2-Ethanol may be dueto the attraction of ethanol in extracting fat content
in the cocoa bean. Based on study from Saldana etal. (2002), they found supercritical carbon dioxide
without the use of co-solvent does not able to extract
theobromine from cocoa seed. Li and Hartland(1996) also showed that it is difficult to extracteither xanthines or cocoa butter from cocoa nibs
using carbon dioxide. There was significant value
from 0.10% to 11.91% of xanthines and 0.06% and1.45% of cocoa butter was obtained from 0 to 23%
of ethanol added in supercritical CO2 system. Similar
to this study, SCO2 was significantly have a lower
cocoa butter obtained compared to SCO2-Ethanol.
The solubility of ethanol can be markedly enhanced
compared with SCO2 under similar condition.
Kopcak and Mohamad (2005) showed thatusing ethanol or iso-propanol as co-solvent withsupercritical fluid increased the yield of caffeine
extraction. Salajegheh et al. (2013) reported that thequantity of oil obtained from whole cocoa beans by
SCO2 was too low to form a basis for a commercial
extraction process. Therefore, the addition of polar
co-solvent ethanol greatly had enhanced cocoa
butter solubility, thus produce high yield of cocoa
butter from the extraction. Salajegheh et al. (2013)stated that supercritical extraction using ethanol as
co-solvent has a higher yield due to the solubility of
ethanol on carbon dioxide. The extract yield from
USE also had a higher value compared to SE andSCO2. A study from Péres et al. (2006) found thatthe extraction of USE was higher in mass yield of
4.36% at 60 min compared to SE; 0.42%. The contactof ethanol with cocoa beans during the process of
USE may enhanced and accelerated the cocoa butter
extraction from cocoa beans compared to SE andSCO2.

Oxidative analysis
Iodine value (IV) of cocoa butter was
insignificantly (p>0.05) affected by the different
extraction methods (33.97-34.56 g I2/100 g) (Table 1)due to the similarity in the fatty acid composition of theextracted oils. As reported by Bhatnagar and Krishna(2013), the IV of Niger seed was insignificant whenapplied with the different type of solvents (petroleum
ether, hexane, chloroform, acetone, methanol andethanol).
Similar pattern was shown in the Peroxide values(PV) of the cocoa butter (Table 1). The PV for SE,
USE and SCO2-Ethanol (2.20, 1.96 and 2.02 meqO2/kg) was not significantly different (p>0.05) fromothers except for SCO2 (2.49 meqO2/kg). The highvalues of PV from SCO2 occur maybe due to theoxidation by carbon dioxide. Oxidation of CO2 may
cause unwanted effects during extraction. Calvo et al.
(1994) stated that oxidative stability of supercriticalextracted sunflower oil could be enhanced using
ultra-pure CO2 (without oxidation). The lower the PV
will contributed to the better quality of oil.
The level of acid value (AV) in the cocoa butter
was not significantly different (p>0.05) except forSE (1.58 mg KOH/g oil) (Table 1). High temperatureduring the extraction process and period of ethanol
exposed to the cocoa nibs during USE may have
either separated lots of fatty acid from glycerol
molecules thus accelerate the hydrolytic rancidity.
The level of AV value was higher in SCO2 and SCO2Ethanol also might be caused by oxidation of CO2
thereby produced water vapour that increases thehydrolytic rancidity (Crowe et al., 2002).
Phytosterols content
Table 2 shows the phytosterols content in cocoa
butter from three different extraction methods. The
campesterol content in the extract using SCO2 andUSE was not significantly higher (p>0.05) (2001Roiaini et al./IFRJ 23(1): 47-54

51

Table 2. Phytosterols content in cocoa butter using different extraction method

Results were expressed as means ± SD for triplicates measurement. Means in the same column with different
letters were significantly different (ρ < 0.05).

and 2030 μg/g of extract) compared to SE andSCO2-Ethanol (1911 and 1925 μg/g of extract).
Nevertheless, no significant difference (p>0.05) was
found for stigmasterol content among SE, USE andSCO2 in extracted cocoa butter except for SCO2Ethanol. On the other hand, beta-sitosterol content
was significantly higher (p<0.05) in SCO2-Ethanol,
followed by SCO2 and no significant difference
was found between SE and USE. Total phytosterols
in SCO2-Ethanol (6441 μg/g of extract) was
significantly higher compared to SCO2 (5492 μg/g ofextract), USE (5106 μg/g of extract) and SE (4960μg/g of extract). The solubility of phytosterols in CO2
in the presence of ethanol increases greatly comparedto other extraction methods. Without co-solvent, theseparation of solute in supercritical CO2 from thesolvent will be not easy. Distillation or evaporation
are needed to obtained the desired products andregenerate the solvent (Li and Hartland, 1996).
The total phytosterols content in cocoa butter
using SCO2 was not significant with SE and USE
similar with the study by Bernardo-gil and Grenha(2002). They stated that the contents of free fatty
acids, phytosterols, triacylglycerols and tocopherols
in the hazelnut oil extracted by SCO2 were equivalent
with soxhlet extraction. From the four methods usedin this study, SE method showed the lowest amountof total phytosterols (4960 μg/g of extract) comparedto the others. SCO2-Ethanol method was among thesuitable method in extracting phytosterols due tomoderate pressure and temperature, higher diffusion
coefficient, lower viscosity and surface tension
compared to solvent and able to generate excellent
mass transfer (Wang and Weller 2006). The operating
temperature in both SCO2 and SCO2-Ethanol (60°C)
was much lower than SE method (78°C). Thus,
higer thermal applied to the cocoa beans might
degrade more phytosterols compared to moderatetemperature. The homogenization procedure during
USE with ethanol for a few minutes also may degrade
the phytosterols composition. Chemat et al. (2004)
stated that Scanning electron micrographs (SEM)
had shown the wrecking of cell walls and liberationof cell contents due to great agitation from USE in
the system, thus encourage the ruptured and damaged
the compounds.
Antioxidant assay (AA) and Total phenolic content(TPC)
Figures 1 and 2 shows the results of DPPH assay
and TPC of the cocoa butter from different extraction
methods. From Figure 1, the DPPH assay of cocoa
butter from SCO2-Ethanol and SCO2 extraction show
significant value (p<0.05) (84% and 82.92% DPPH)
compared to SE and USE (44.72% and 37.91%).
The TPC from SCO2 and SCO2-Ethanol was
significantly higher (p<0.05) (29.5 mg GAE/100 g
extract and 28.77 mg GAE/100 g extract) compared
to SE and USE (21.77 mg GAE/100 g extract and14.42 mg GAE/100 g extract). The TPC value was
the highest in SFE-CO2 extracts, but unlikely for
DPPH value. Low DPPH and TPC from SE and USE
method might be due to high thermal involved during
extraction, direct contact of cocoa bean with ethanol
during processing and homogenization of cocoa nibs
in ethanol possibly affected the polyphenolic content.
Arlorio et al. (2008) also stated that various heat-based
treatment steps, pressure or collision of ultrasoundwaves changed the content and the composition ofpolyphenolic proportion of cocoa beans. Azizah et al.
(1999) stated that various causes such as variation of
cocoa, stage of beans mature, the use of solvent andthe technique of extraction influenced the efficiencyof polyphenols extraction. Thus, the application ofdifferent techniques such as different temperature,pressure and level of homogenization in each

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Roiaini et al./IFRJ 23(1): 47-54

Figure 1. DPPH assay for different types of extraction
methods

extensive extraction time and have minimum
efficiency. Furthermore, as the temperature iselevated during the extraction, the natural constituent
may be deteriorating because of their irresistible tothermal heat. Moreover, this method required a longextraction time and large amount of organic solvent.
The requirement of large amount of solvent may
cause additional costs and danger to environmental
due to waste of poisonous solvents. This finding
may be benefits the cocoa industry to produce cocoa
butter with high yield and preserve the bioactive andantioxidant content.
Acknowledgement
This research was supported by Universiti
Putra Malaysia with grant project number GPIPS/2013/9399841.
References

Figure 2. TPC values for different types of extraction
methods

extraction method of cocoa nibs in this study may
deteriorated the phenolic content in different amount.
From the results, high DPPH does not indicate
the presence of phenols in cocoa extracts. Othman
et al. (2007) stated that total antioxidant/antiradical
capacity was influenced by the present of methyl
xanthines (theobromine and caffeine), minor
flavonoids and pigments in the cocoa extracts. The
solubility of other compounds in water, ethanol andother solvents also contribute to the high antioxidant
content other than phenolics.
Conclusion
The different extraction methods significantly
affected the yield, phytosterols and antioxidant
content of cocoa butter. The SFE with co-solvent
able to extract more cocoa butter, higher yield ofphytosterols and antioxidant compared to othermethods. However, their oxidative value does notaffected by extraction methods due similar types of
cocoa bean used in study. Carbon dioxide present in
the SCO2 promotes oxidation of the oil extract. The
SCO2-Ethanol is much simpler and more effective
than SE, USE and SCO2 method in obtaining highphytosterols and antioxidant content. SE was notrecommended for extraction because it requires

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