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Methodology
Nor Faadila M. Idrus1, Noor A. Febrianto1, Wahidu Zzaman1,2, Tang E. Cuang1 and Tajul A. Yang1*
1
School of Industrial Technology, Food Technology Division, Universiti Sains Malaysia, Penang 11800, Malaysia
2
Department of Food Engineering and Tea Technology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
Virgin coconut oil is a functional food which provides health benefits. It is different from other cook-
ing oils in that it has medium-chain fatty acids rather than long-chain fatty acids. The traditional aqueous
extraction of the oil is very flexible that can be applied in a medium to large-scale industry. The study was
carried out to optimize the aqueous extraction of virgin coconut oil using response surface methodology.
The most influencing three factors were used for this experiment, which are the coconut milk-to-water
percentage, fermentation and refrigeration time. The optimization study showed that the method can pro-
duce the best yield with quality by using coconut milk (73.8%), fermented (14.1 h) and refrigerated time
(20.5 h). Coconut milk percentage and fermentation time significantly affected the response of extraction
yield (p ≤ 0.01). The aqueous extraction can be used commercially for the production of virgin coconut oil
as the method is environmental friendly.
Keywords: virgin coconut oil, aqueous extraction, optimization, response surface methodology
Introduction Unlike the common coconut oil that produced from co-
Today, the trend of oil consumption has shifted from the pra, which generally had been long stored, and currently
use of purified oil into natural oil. The development of health produced at medium and large industries using specific
issues and the increasing awareness of chemical free product equipment whereas VCO is usually obtained through wet
are several reasons that initiate this phenomenon. This condi- processing (aqueous) from fresh coconut, which is possible
tion has raised the growing market of less-proceeded oil such to be applied at small and even at home kitchen scale. Ma-
as virgin olive oil, virgin sunflower seed oil and virgin coco- rina et al. (2009) had described several methods of VCO ex-
nut oil (VCO). Currently, VCO is used as directly-consumed traction, via traditional wet extraction; chilling, freezing and
supplement due to its nutritious components such as high thawing techniques; fermentation by microorganism culture
content of medium fatty acid and shows good digestibility and enzymatic extraction.
(Cheman et al., 1997). Hypocholesterolemic and antioxidant The simple process and no specific equipment needed
effect of some unsaponifiable components, such as vitamins, is the advantage of traditional aqueous extraction compared
polyphenols, sterols is effective in reducing the lipid levels to other extraction methods (Mechanical pressing, solvent
and lipid peroxidation (Anderson et al., 2001; Khor et al., extraction, etc.). Traditional wet extraction can be carried out
1998; Nicolosi et al., 2001). It is also reported that consump- using water to extract oil from the pulp of fresh grated coco-
tion of coconut oil can lower body-fat deposition and reduce nut. This process involved the step of squeezing the grated
blood clotting tendency (Bruce-Fife, 2001). Thus, the less coconut flesh to obtain coconut milk and then followed by
processed VCO is preferred because it neglects the common separation of coconut milk into oil and non-oil product. In
process of refining, bleaching and deodorizing that could re- common practice, the destabilization of coconut milk emul-
move the useful minor constituent (McWilliams, 2001). sion to separate the oil from the mixture (Marina et al., 2009)
can be done in several ways. Suhardiyono (1988) mentioned
*To whom correspondence should be addressed. that it can be done by cooking the coconut milk for 3 − 4 h
E-mail: taris@usm.my to release the water through evaporation, whereas Marina
730 N. F. M. Idrus et al.
ment performed using a combination of the variables for fatty acid value, acid value, peroxide value, K232, and K270
six response functions (yield, FFA, AV, POV, K232 and K270) the sum of squares of the sequential model were analyzed
is shown in Table 1. The data were analyzed by multiple together with the corresponding fitting of the explanatory
regressions to fit the quadratic equations to the dependent models. The sequential model of sum of squares analyses
variables. Statistical analysis was performed using RSM soft- indicated that adding terms up to quadratic significantly im-
ware, Design-Expert 8 (Stat-Ease Inc, Minneapolis, USA). proved the model (p < 0.05) (Table 2) with the exception of
Analysis of variance (ANOVA) was performed to evaluate K270 (0.0602). Koocheki et al. (2009) mentioned that coeffi-
the adequacy of the generated mathematical models. In this cients of determination (R2), adj-R2 and coefficients of varia-
study, Box-Behnken statistical screening design was used to tions (CV) also indicate the adequacy of the model, which R2
optimize and evaluate main effects, interaction effects, and should not be less than 80% to ensure the suitability of the
quadratic effects of variables. model. In this study, the R2 values for extraction yield, FFA,
The nonlinear computer-generated quadratic model is AV, POV, K232 and K270 were 0.951, 0.954, 0.953, 0.979, 0.983
given as: and 0.967, respectively (Table 3).
All responses have shown the adj-R2 values higher than
Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3
0.80 to ensure the model adequacy, whereas the coefficient
+ b23X2X3 + b11X12 + b22X22 + b33X32
of variation of all the responses were lower than 10%. Coef-
Where Y is the measured response associated with each fac- ficient of variation (CV) measures the dispersion of a prob-
tor level combination; b0 is an intercept; b1 to b33 are the re- ability distribution, which high CV will indicate high varia-
gression coefficients; and X1, X2, and X3 are the independent tion in the mean value and sufficient response model cannot
variables. be developed. Thus, CV should not be higher than 10%
Numerical optimization technique of the Design-Expert (Koocheki et al., 2009). Moreover, the adequacy of the qua-
software was used for simultaneous optimization of the dratic model was convinced with the non-significant effect of
multiple responses. The desired goals for each variables and lack of fits.
response were chosen. All the independents variables were Yield Coconut milk percentage, fermentation time and
kept within range while the responses were either maximized their quadratic effects significantly affected the response of
or minimized. extraction yield (p ≤ 0.01). The quadratic effect of refrigera-
tion time and the interaction effect of coconut milk percent-
Results and Discussion age and refrigeration time affected the experimental results
Model fitting The variation of the extraction yield, free less significantly (p ≤ 0.05) (Table 3). The regression equa-
Table 1. Experimental design and analysis result in coded (x) and actual level (X) of variables.
Table 2. Sequential model sum of square of yield (Y1), FFA (Y2), AV (Y3), POV (Y4), K232 (Y5) and K270 (Y6).
Table 3. Condensed ANOVA table for yield (Y1), FFA (Y2), AV (Y3), POV (Y4), K232 (Y5) and K270 (Y6).
Y1 Y2 Y3 Y4 Y5 Y6
Source of
variations Sum of p-value Sum of p-value Sum of p-value Sum of p-value Sum of p-value Sum of p-value
square square square square square square
Model Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic
Constant 41.8900 0.0008 0.1100 0.0007 0.3900 0.0007 0.6500 <0.0001 0.0100 <0.0001 0.0014 0.0002
X1 4.2300 0.0076 0.0015 0.2002 0.0061 0.1835 0.0200 0.0168 0.0003 0.0083 0.0001 0.0038
(F)X2 11.8800 0.0004 0.0110 0.0062 0.0380 0.0078 0.0700 0.0006 0.0062 <0.0001 0.0004 0.0002
(R)X3 0.1200 0.5565 0.0091 0.0104 0.0330 0.0111 0.3400 <0.0001 0.0026 <0.0001 0.0007 <0.0001
X1X2 0.0160 0.8284 0.0004 0.4907 0.0012 0.5279 0.0057 0.1419 0.0002 0.0369 0.0000 0.0260
X1X3 2.4200 0.0265 0.0006 0.3936 0.0020 0.4215 0.0510 0.0016 0.0004 0.0071 0.0000 0.3794
X2X3 0.0140 0.8351 0.0064 0.0227 0.0200 0.0326 0.0630 0.0009 0.0000 0.6361 0.0002 0.0015
X12 15.4400 0.0002 0.0790 <0.0001 0.2900 <0.0001 0.0420 0.0027 0.0004 0.0065 0.0000 0.3415
X22 4.6600 0.0060 0.0001 0.6808 0.0007 0.6546 0.0320 0.0054 0.0004 0.2385 0.0000 0.0254
X32 3.2400 0.0142 0.0012 0.2516 0.0033 0.3118 0.0320 0.0054 0.0000 0.9454 0.0000 0.1379
Lack of Fit 1.7200 0.0718 0.0042 0.0777 0.0160 0.0514 0.0094 0.1985 0.0000 0.7066 0.0000 0.0877
R2 0.951 0.954 0.959 0.979 0.983 0.967
Adj-R2 0.888 0.896 0.892 0.951 0.960 0.924
CV 1.940 5.250 5.300 8.200 0.440 7.100
tion for the response functions in the actual level of this vari- of water added for the extraction of oil. Che Man et al. (1997)
able is: reported that adding less water contributed to a higher pro-
portion of oil, but if the coconut content is too high or water
Y1 = 10.8325 + 0.4315X1 + 0.47802X2
content is too low, yield will be decreasing.
− 0.003064X12 − 0.007309X22 + 0.00609X32
Generally, the longer fermentation time will give higher
The significant quadratic effect of coconut milk percent- yield. Waisundara et al. (2006) evaluated the effect of micro-
age and fermentation time indicates that yield extraction bial lipase that may have been produced due to the growth
increased with the increase in coconut milk content and of microorganism during storage at room temperature. The
fermentation time, and decreased at certain range (Fig 1). enzymes will degrade the polysaccharides and improve the
For coconut milk percentage, increasing coconut content yields. Suitable environmental condition such as acidic en-
or decreasing water content will increase the yield. As the vironment, room temperature and the presence of catalysts
percentage of water added was increased, the oil content de- (iron) will optimize the lipase reaction (Ibrahim, 1994). Pre-
creased. Higher proportions of water increased the dilution cipitation of soluble protein in the interfacial film occurred as
effect and therefore decreased the oil yield in coconut cream a result of lactic acid formation during fermentation. Lactic
(Che Man et al., 1997). Banzon et al. (1990) found that the acid destabilized the protein and caused water to be released
composition of coconut cream is largely based on the amount (Suhardiyono, 1992). Besides, the separation of a mixture of
Optimization of the Aqueous Extraction of Virgin Coconut Oil 733
Fig. 1. Left: Response surface for the effect of coconut milk percentage and fermentation time on the yield of VCO at constant refrigeration
time 24 h. Right: Response surface for the effect of refrigeration time and fermentation time on the yield of VCO at constant coconut milk
percentage 50%
Fig. 2. Left: Response surface for the effect of coconut milk percentage and refrigeration time on the free fatty acids of VCO at constant
fermentation time 24 h. Right: Response surface for the effect of coconut milk percentage and fermentation time on the acid value of VCO
at constant refrigeration time 24 h.
water and protein-fat will be more complete if the fermen- tions in the actual level of this variable are:
tation time is longer, so the yield will be higher. However,
the lipase is impaired after the reaction reached a maximum Y2 = 1.4 + 0.00142X2 + 0.000218X12
level (Frazier and Westhoff, 1994). Y3 = 2.675 + 0.002577X2 + 0.0004168X12
In this experiment, refrigeration was utilized to separate
the oily layer and non-oily layer of solution and the longer FFA and AV are dependent on coconut milk percentage
refrigeration time decreased the yield (Fig 1). The decreasing and decreased with the increase in coconut milk percentage
yield as the increase of refrigeration time currently correlated but an inflection point occurred at certain range (Fig 2). High
with the percentage of coconut milk to water used. At higher coconut milk content with low water content has a negative
proportion of water used, the tendency of yield is decreasing effect to FFA and AV value until the inflection point where
as the longer refrigeration time, vice versa, at lower propor- it becomes contrariwise. This is because hydrolysis liberates
tion of it the yield is increasing as the longer refrigeration the FFA from the parent oil and thus releasing the FFA that
time. are responsible for the rancid aroma (Freeland-Graves and
FFA and AV From Table 3, the variation in fermenta- Peckham, 1996). Hydrolytic rancidity is a result of a break-
tion time and the quadratic effect of coconut milk percentage ing of the glyceride molecule at the ester linkage, forming
show the most significant effect (p ≤ 0.01) on the acid value, fatty acids which give out undesirable odor (Brien, 1998).
whereas the linear effect of refrigeration time and its interac- The presence of large amounts of water in the coconut milk
tion with fermentation time were less significant (p ≤ 0.05). promotes faster microbial growth and the enzyme will react
The regression equation for the FFA and AV response func- more actively. The higher the moisture content in the oil
734 N. F. M. Idrus et al.
resulted in the higher percentage of FFA (Che Man et al., bination of high coconut milk percentage and longer time of
1997). refrigeration produce lower POV; whereas the use of longer
Increases in refrigeration time affect the increase in FFA fermentation time also could produce lower POV.
and AV (Fig 2). Cancel (1979) reported that the amount of K232 and K270 specific extinction coefficients K232 is a
fatty acid produced will be decreased if higher temperature spectrophotometric measure of conjugated dienes, which is
involved in the extraction. Types and activities of enzymes used along with the peroxide index to evaluate the primary
also will be affected, such as enzyme lipase which brings oxidation of the oil. K270 is used to evaluate, spectrophoto-
about an increased release of fatty acids from oil during metrically, the conjugated trienes and indicate the state of
processing at lower temperature (Chen and Diosady, 2003). secondary oxidation of the oil (Torres, 2006). The presence
The FFA of the samples increased constantly with increase in and formation of conjugated dienes and trienes would pre-
storage time. This might be due the production of microbial dispose the oil for further oxidative changes (Subramanian et
lipase during storage (Waisundara et al., 2006). al., 1998).
The significant linear effect of fermentation time indi- From Table 3, the K232 response analysis showed that it
cates that FFA and AV increased with the increase in fermen- was significantly affected by fermentation time, its quadratic
tation time. Longer fermentation time gives higher FFA and effect, its interaction with refrigeration time, the variation in
AV. The lipase released by the microorganism during fer- refrigeration time and fermentation time (p ≤ 0.01). The in-
mentation will catalyze the action of moisture to hydrolyze teraction effect of coconut milk percentage and fermentation
the triglycerides (Brien, 1998). time affected the experimental results less significantly (p ≤
POV POV generally indicates the extent to which an oil 0.05). The regression equation for the response functions in
sample has undergone primary oxidation (Raghavendra and the actual level of this variable is:
Raghavarao, 2011). In this experiment, the value varied from
Y5 = 1.15013 − 0.001242X1 + 0.002687X2 + 0.0006187X3
0.25 − 1.00 meq/kg oil. This result still in agreement with
− 0.00002166X1X2 – 0.00003166X1X3 + 0.00001508X12
the result presented by Raghavendra and Raghavarao (2011)
which found that the peroxide value of VCO was around Increase in coconut milk percentage increased the value
0.81 ± 0.02, whereas commercial VCO was 0.94 ± 0.02. We of K232 at short refrigeration time, but increased the value of
found that POV was significantly affected by almost all of K232 at long refrigeration time (Fig 4). There are not so much
the linear, interaction and quadratic effect of coconut milk changes of K232 value at low refrigeration time compared to
percentage, fermentation time and refrigeration time (p < high refrigeration time. Oxidation process increased with
0.05) except the interaction of coconut milk percentage and increasing moisture content (Akinoso et al., 2010). At high
fermentation time (p = 0.1419). The regression equation for refrigeration time, increasing coconut milk percentage (de-
the response functions in the actual level of this variable is: creasing moisture content) will decrease the value of K232. At
short refrigeration time, K232 did not change much with the
Y4 = 0.2 + 0.032X1 − 0.00885X2 − 0.039X3 − 0.000375X1X3
coconut milk percentage. This might be due to initial storage
– 0.000868X2X3 − 0.00016X12 − 0.000607X22 − 0.000607X32
in the refrigerator which the oxidation rate is still low.
Coconut oil is mainly consisted by saturated fatty acid The significant linear effect of refrigeration time percent-
that more resistant to oxidation due to it less reactivity age indicates that K232 value decreased with the increase in
over influence of light, heat and oxidation (Francis, 1999). refrigeration time. Alonso et al. (2005) mentioned that lipid
Raghavendra and Raghavarao (2011) mentioned that virgin oxidation occurs fairly slowly at low temperature. The effect
coconut oil contain 43 − 53% lauric acid, 16 − 21% myristic of decreasing in K232 value is more obvious if the refrigera-
acid, 7.5 − 10% palmitic acid, 5 − 10% caprylic acid, and tion time is longer. Due to the separation of water from but-
4.5% − 5.8% capric acid as major constituent. In spontane- ter fat phase is more complete, therefore moisture content in
ous oxidation, radical oxygen will attack the unsaturated the oil is lower, giving lower oxidation rates. Theoretically,
bonds in unsaturated fatty acid to produce hydroxyperoxide coconut oil should show a low rate of oxidation because it
(Ketaren, 1986). Raghavendra and Raghavarao (2011) also contains low levels of unsaturated fatty acids (Che Man,
mentioned that VCO was also consisted of 5 − 10% oleic 1997).
acid (C18:1) and 1.0 − 2.5% linoleic acid (C18:2) which sus- As shown in Table 3, coconut milk percentage, refrigera-
ceptible to be oxidized. Moreover, during fermentation and tion time, fermentation time, interaction effect of refrigera-
refrigeration in his experiment, the container containing a tion time and coconut milk percentage significantly affected
mixture of coconut milk and water was left open-air and the response of K270 (p ≤ 0.01). The interaction effect of
unprotected from ambient light. According to Fig 3, the com- coconut milk percentage between fermentation time, and
Optimization of the Aqueous Extraction of Virgin Coconut Oil 735
Fig. 3. Left: Response surface for the effect of coconut milk percentage and fermentation time on the peroxide value of VCO at constant re-
frigeration time 24 h. Right: Response surface for the effect of coconut milk percentage and refrigeration time on the peroxide value of VCO
at constant fermentation time 24 h.
Fig. 4. Left: Response surface for the effect of coconut milk percentage and fermentation time on the K232 of VCO at constant refrigeration
time 24 h. Right: Response surface for the effect of coconut milk percentage and refrigeration time on the K270 of VCO at constant refrigera-
tion time 24 h.
fermentation time between refrigeration time and quadratic tion and refrigeration, container containing mixture of coco-
effect of fermentation time affected the experimental results nut milk and water is left open-air and is not wrapped with
less significantly (p ≤ 0.05). The regression equation for the aluminum foil. So with the presence of light, a significant
response functions in the actual level of this variable is: increase of K270 was noticed to be faster at the beginning and
then progressively decreased because of the small permeabil-
Y6 = 0.024875 + 0.000558X1 – 0.00085X2 – 0.000808X3
ity of the means (Vekiari et al., 2007). Caponio et al. (2005)
− 0.0000125X1X2 + 0.00004687X2X3 + 0.00002552X22
reported that oils kept under light contained products of sec-
K270 value is dependent on coconut milk percentage and ondary oxidation.
decrease with the increase in coconut milk percentage (Fig CIE- L*a*b* Color From the result in Table 4, hue
4). The increase in oxidation ensured by the increase in K270 angle of all samples are in the range of 95 − 97°, which is the
value due to increasing number of compounds resulting from area within 90° and 180° is represented by greenish-yellow
peroxide degradation (Vekiari et al., 2007). The result showed color. But due to low C* value, indicating low color inten-
that rate of secondary oxidation was too low. This can be sity, thus the color is clear or water-like color. Meanwhile,
proven that virgin coconut oil produced is very stable against hue angle of reference coconut oil was 109.69°, which is in-
oxidation due to the high degree of saturated fatty acid. dicated by light-greenish color. But for the same reason, low
The significant linear effect of refrigeration time shows C* value giving water-like color. The total color difference
that K270 value increased with the increase in refrigeration (ΔE) values of all VCO samples were just in the range of 1.56
time (Fig 4). Increase in fermentation time also induced a − 2.31, meaning that the color of the samples is quite similar
partially exponential increase in K270 value. During fermenta- to the reference oils, consumers hardly differentiate them.
736 N. F. M. Idrus et al.
Table 4. Experimental result for the response function for Acknowledgments We gratefully acknowledge the Fellowship
CIE-L*a*b* color. Scheme of the Institute of Postgraduate Studies, Universiti Sains
Expt. Malaysia (Grant No. 1001/PTEKIND/814141) and anonymous ref-
L* a* b* C* Hue ∆E
No. erees for comment and constructive advice in improving this manu-
1 98.92 −0.31 3.05 3.07 95.74 1.95 script.
2 99.37 −0.29 2.85 2.86 95.81 2.28
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4 99.29 −0.30 2.82 2.84 96.07 2.20
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