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Industrial Crops and Products xxx (2016) xxx–xxx

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Optimization of neutralization parameters in chemical refining of

kenaf seed oil by response surface methodology
Sook-Chin Chew a , Chin-Ping Tan b , Kar-Lin Nyam a,∗
a
Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, 56000 Kuala Lumpur, Malaysia
b
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Kenaf seed oil has been suggested to be used as edible oil due to its high nutritional value. Optimal
Received 18 August 2016 parameters in the refining process for kenaf seed oil are still limited in order to produce edible kenaf seed
Received in revised form 9 November 2016 oil with high quality attributes. Response surface methodology with central composite design was used
Accepted 21 November 2016
to study the effect of the parameters in the neutralization stage of chemical refining process, namely the
Available online xxx
excess level of sodium hydroxide (0–5%), temperature (25–85 ◦ C) and time (10–50 min). The experimental
data obtained from the twenty experimental runs were fitted to a second-order polynomial equation
Keywords:
using multiple regression analysis. Well-fitting models were successfully generated for the responses
Neutralization
Kenaf seed oil of free fatty acids (R2 = 0.9954) and peroxide value (R2 = 0.9365) with the probability value less than
Temperature 0.0001, which demonstrated a high significance for the regression models. The optimum parameters
Time were recommended at an excess level of sodium hydroxide of 3.75%, temperature of 40 ◦ C and time of
Free fatty acids 20 min to provide the neutralized kenaf seed oil with minimizing free fatty acids and peroxide value.
Peroxide value Under these parameters, free fatty acids of 0.12% and peroxide value of 1.57 meq/kg were obtained in the
neutralized kenaf seed oil.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Kenaf (Hibiscus cannabinus L.) is an herbaceous annual crop from
the Malvaceae family, which is particularly valuable for its fiber
content to be used as making ropes, sacks, and carpets. Kenaf is
also cultivated for new applications in pulp and paper industry, oil
absorption and potting media, board production, filtration media
and animal feed (Ayadi et al., 2011). Kenaf is represented as an
alternative fiber crop to meet the strong demand for fiber and for-
age industries due to kenaf is a fast-growing annual plant, which
can help to reduce massive deforestation. Kenaf plant is cultivated
in more than 20 countries and the total production of kenaf and
other allied plants is 284 100 tons in 2010/2011 (International Jute
Study Group, 2012). Kenaf is mainly produced in India, China, and
Thailand (Chan and Ismail, 2009).
Malaysia government has changed National Tobacco Board to
National Kenaf and Tobacco Board in order to take the strategic of
stimulating the development of kenaf industry (Wong et al., 2014).
Abbreviations: RSM, response surface methodology; ANOVA, analysis of vari-
ance; FFA, free fatty acids; PV, peroxide value.
∗ Corresponding author.
E-mail address: nyamkl@ucsiuniversity.edu.my (K.-L. Nyam).
There was an increasing annually from 7190 kg of kenaf seeds pro-
duced in 2006 to 151 250 kg of kenaf seeds in 2011 in Malaysia
(Ishar, 2012). Seeds represent a potential source of proteins and
lipids with a high nutritional value. Nyam et al. (2009) reported that
kenaf seeds contain 9.1% moisture, 21.8% crude protein, 13.6% crude
fiber, 5.9% ash, and 28.8% total carbohydrate. Besides, kenaf seeds
contain 20.8% of oil, which is a rich source of unsaturated fatty acids,
including oleic (33%) and linoleic (32.2%) acids (Nyam et al., 2009;
Chew et al., 2016). Unsaturated fatty acids help to reduce the major
cardiovascular risks such as hyperlipidemia, hypercholesterolemia,
and hypertriglyceridemia (Makni et al., 2008; Mariod et al., 2009).
Ng et al. (2015) proved that kenaf seed oil contains compara-
ble anti-hypercholesterolemic effect with simvastatin (commercial
hypocholesterolemic drug). Besides that, polyphenols, tocopherols,
and phytosterols are highly exhibited in the kenaf seed oil (Chew
et al., 2016). Thus, kenaf seed oil has been suggested to be used
potentially as a source of edible oil due to its unique composition
and nutritional value. Besides, kenaf seed oil has been suggested
to be used as a possible biodiesel feedstock in previous studies
(Rathana et al., 2010; Jindal and Goyal, 2012).
Oil produced by mechanical pressing or, more often, by solvent
extraction from the raw materials is termed crude oil. Currently,
research that focused on the functional properties and applica-
tions of crude kenaf seed oil has been increased. Kenaf seed oil

http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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Please cite this article in press as: Chew, S.-C., et al., Optimization of neutralization parameters in chemical refining of kenaf seed oil by
response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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has been studied by using microencapsulation and nanoemulsions
approaches to improve its bioavailability in order to develop a func-
tional product that beneficial in cosmetic, food, nutraceutical and
pharmaceutical industries (Chew et al., 2015; Cheong et al., 2016).
However, refining process, which including the stages of degum-
ming, neutralization, bleaching and deodorization is required to
carry out in order to remove the undesirable components in the
crude oil and to produce odorless, bland and oxidative stable oils,
which are acceptable to consumer (Farhoosh et al., 2009). The valu-
able bioactive compounds are easily removed during the refining
process. Thus, vegetable oil manufacturer is forced to optimize the
refining process in order to maximize the removal of impurities and
minimize the losses of bioactive compounds during the refining
process due to the market competition.
Chew et al. (2016) reported that chemical refining process
improves the quality of kenaf seed oil. However, the refining con-
ditions can be studied in order to optimize the refining conditions
to get refined kenaf seed oil with high-quality attributes. Chemical
refining has fewer effects on the desirable bioactive compounds
in the oil compared with physical refining (Suliman et al., 2013).
Chemical refining has been widely used in the processing of veg-
etable oil and reported to improve the quality attributes of wheat
germ oil (Wang and Johnson, 2001), sunflower oil (Suliman et al.,
2013), soybean oil and canola oil (Farhoosh et al., 2009) in the
previous studies. Neutralization is a stage where an appropriate
amount of NaOH is added with some excess percentages, to neu-
tralize the free fatty acids in the crude oil, results in the formation
of sodium soaps in oil (Dumont and Narine, 2008). Free fatty acids
are required to be removed in the neutralization stage as it can act
as pro-oxidants in the oil to accelerate the rate of hydroperoxide
decomposition (Ghazani and Marangoni, 2013).
Against this background, the present study was proposed to
optimize the parameters in the neutralization stage of the refining
process by using response surface methodology. Response surface
methodology (RSM) is a powerful statistical tool used to predict
the optimum experimental conditions and to describe the rela-
tionship between independent variables based on one or more
responses. Process optimization is applied through RSM with a
reduced number of experimental trials (Pinela et al., 2016). The
effects of different parameters during the neutralization stage, such
as excess level of NaOH, temperature and time were investigated
based on the responses of free fatty acids and peroxide value in
kenaf seed oil.
2. Materials and methods
2.1. Solvent extraction of kenaf seed oil
Kenaf (Hibiscus cannabinus L.) seeds were obtained from
Malaysia Agricultural Research and Development Institute
(MARDI), Selangor, Malaysia. The kenaf seeds were ground into
fine powder using a food grinder (Panasonic, Japan). Soxhlet
extractor was used to extract the crude oil from the seeds at 60 ◦ C
for 3 h by using hexane as solvent. Hexane was evaporating off by
using Buchi Multivapor P-6 at 55 ◦ C under a reduced pressure of
241 mbar and followed by flushing with 99.9% nitrogen to recover
the oil.
2.2. Degumming of kenaf seed oil
The degumming process for kenaf seed oil was carried out
according to a previously described method (Chew et al., 2016).
The parameters were optimized in the preliminary trial. Phospho-
ric acid (85% concentration) (0.09% w/w) was added to the crude
kenaf seed oil in a water bath at 40 ◦ C, and the mixture was agitated
Please cite this article in press as: Chew, S.-C., et al., Optimization of neutralization parameters in chemical refining of kenaf seed oil by
response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
Table 1a
Coded and actual levels of independent variables used for central composite design
in the neutralization of kenaf seed oil.
Symbol Variables Levels
−2 −1 0 +1 +2
X1 Excess level of NaOH (%) 0 1.25 2.50 3.75 5.00
X2 Temperature (◦ C) 25 40 55 70 85
X3 Time (min) 10 20 30 40 50
at 175 rpm for 10 min. Then, 22.4% of distilled water was added into
the oil and followed by agitation at 175 rpm for 30 min. The mix-
ture was centrifuged at 10 000 × g for 15 min in order to separate
the gums.
2.3. Neutralization of kenaf seed oil
After determining the free fatty acids content in the degummed
oil, a stoichiometric quantity of NaOH solution (16◦ Be, 11.6% w/w)
was added with an excess level (X1 ) that varied from 0 to 5% in
order to neutralize the free fatty acids. The mixture was heated at a
temperature (X2 ) ranged from 25 to 85 ◦ C under constant stirring at
1000 rpm for a time (X3 ) that varied from 10 to 50 min. The neutral-
ized oil was washed three times with ultra-pure water (15% w/w),
and the residual soap was removed by centrifugation at 10 000 × g
for 10 min.
2.4. Experimental design and statistical analysis
In this study, a design of experiment including three factors,
specifically excess level of NaOH (%), temperature (◦ C) and time
(min) was decided. Five levels were applied to each of the three
factors as introduced in Table 1a. With the help of Design Expert
version 9.0 software (State-Ease, Inc.), RSM with a central compos-
ite design with an axial value of 2 was employed. This design of
experiment required 20 experimental runs, which consisted eight
(23 ) factorial points, six extra points (star points) and six center
points. The six replicates for the center point were used to evalu-
ate the experimental error and calculate the repeatability of the
method (Nyam et al., 2010). All these conditions were selected
based on preliminary experiments. The responses to minimize
were free fatty acids (%) and peroxide value (meq/kg) of the neu-
tralized oil. The experimental data was fitted to the second-order
polynomial model to obtain regression coefficients. The general-
ized second-order polynomial model used in the response surface
analysis as follows:
  
Y = B0 + Bi X i + Bii Xi 2 + Bij Xi Xj
where Y represents the experimental response, B0 is a constant,
Bi , Bii and Bij are the linear, quadratic, and interactive regression
coefficients of the model, and Xi and Xj are independent variables
in coded values.
The experimental runs were randomized in order to reduce the
effects of the unexpected variability in the observed responses. Lack
of fit, the coefficient of determination (R2 ) and the F-test value
obtained from the analysis of variance (ANOVA) were used to eval-
uate the adequacy of the developed model. Regression analysis and
response surface contour plots were generated to determine opti-
mum conditions and analyze how the interactions of independent
variables affected the overall response. The significance level was
based on a confidence level of 95.0%. Verification of model valid-
ity was confirmed by comparing the experimental data with the
predicted results of the optimized model.
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Table 1b
Experimental data and the observed response values with different combinations of excess level of NaOH (X1 ), temperature (X2 ) and time (X3 ) in the neutralization of kenaf
seed oil.

Std Order Run Order Coded variable Actual variable values FFA (%) PV (meq/kg)

X1 X2 X3 X1 X2 X3

6 1 1 −1 1 3.75 40 40 0.15 2.4

9 2 0 0 0 2.50 55 30 0.18 2.4
10 3 −1 1 1 1.25 70 40 0.66 2.4
7 4 0 0 0 2.50 55 30 0.18 2.2
4 5 1 1 −1 3.75 70 20 0.14 1.8
1 6 −1 −1 −1 1.25 40 20 0.48 2.6
5 7 0 0 0 2.50 55 30 0.16 2.2
3 8 −1 1 −1 1.25 70 20 0.63 2.5
12 9 −1 −1 1 1.25 40 40 0.49 2.3
2 10 0 0 0 2.50 55 30 0.21 2.4
8 11 1 1 1 3.75 70 40 0.16 2.2
11 12 1 −1 −1 3.75 40 20 0.14 1.4
20 13 0 0 −2 2.50 55 10 0.16 2.0
17 14 2 0 0 5.00 55 30 0.16 1.4
14 15 0 2 0 2.50 85 30 0.32 2.7
19 16 0 0 0 2.50 55 30 0.17 2.3
18 17 0 −2 0 2.50 25 30 0.15 2.3
13 18 0 0 2 2.50 55 50 0.17 2.4
15 19 0 0 0 2.50 55 30 0.16 2.3
16 20 −2 0 0 0 55 30 1.02 2.3

2.5. Determination of free fatty acids (FFA) 3. Results and discussion

Determination of FFA was carried out by using AOCS Official
Method (2000) and expressed as oleic acid in percentage.
2.6. Determination of peroxide value (PV)
The PV was analyzed using AOCS Official Method (1998) and
expressed as milliequivalent per kg oil (meq/kg).
2.7. Oil quality analysis and fatty acid profile
Oil quality analyses were carried out in the crude and neutral-
ized kenaf seed oil by using the optimized parameters. Refractive
index was determined according to method Cc-7-25 of AOCS (1989)
and specific gravity was measured by the ratio of the mass of 10 mL
of oil in pycnometer to the mass of 10 mL of Milli-Q water. PV (),
FFA (AOCS, 2000), and iodine value (IUPAC, 1987) was analyzed
in the oil sample. Fatty acid profile in the oil sample was ana-
lyzed according to the previously established method of Nyam et al.
(2009) and Chew et al. (2016). The FAME was prepared by using
50 mg of oil and followed by adding 950 ␮L of n-hexane and 50 ␮L
of sodium methoxide in methanol (30 mL/100 mL). The top layer
(1 ␮L) was injected into a gas chromatography (Agilent Technolo-
gies 7890A, USA) coupled with a flame ionization detector and a
polar capillary column BPX70 (0.32 mm internal diameter, 30 m
length and 0.25 ␮m film thickness; SGE International Pty. Ltd., Vic-
toria, Australia), with a split ratio of 50:1. Oven temperature was
programmed from 115 to 180 ◦ C at 8 ◦ C/min and hold for 17 min
and then programmed to 250 ◦ C at 20 ◦ C/min. Individual fatty acids
were identified by comparing the retention times with a certified
fatty acid methyl esters (FAME) mix (Sigma-Aldrich, St. Louis, USA),
which was analyzed under the same operating conditions. The fatty
acid composition was expressed as relative area percentage. All
analyses were carried out in triplicate. The results were analyzed
using MINITAB 16 (Minitab Inc, Pennsylvania, USA) by independent
T-test and the difference was considered statistical significantly at
the level of p-value < 0.05.
3.1. Fitting the models
Table 1b shows the twenty experimental data sets were
analyzed by multiple regression analysis and ANOVA. Central com-
posite design was used to optimize the neutralization parameters
(excess level of NaOH, temperature and time) for reducing the FFA
and PV in neutralized kenaf seed oil. The FFA of neutralized kenaf
seed oil obtained in this study ranged from 0.14 to 1.02%, while
PV of neutralized kenaf seed oil ranged from 1.4 to 2.7 meq/kg.
Determination of FFA is important to evaluate the efficiency of
the neutralization stage to neutralize the FFA. Insoluble soaps are
formed by the reaction of FFA with NaOH solution that can be easily
separated through centrifugation. The neutralization stage can be
effectively reduced FFA contents in the oil.
The best-fitting models were generated through multiple linear
regressions with backward elimination to remove the insignifi-
cant terms (p > 0.05) from the suggested quadratic model to allow
improvement of the models. Tables 2 and 3 show the ANOVA results
for the reduced quadratic models for the responses of FFA and PV in
neutralized kenaf seed oil. It can be seen that there was a high sta-
tistically significant multiple regression relationships between the
independent variables and the response variables. The probability
(p) values of the regression models for FFA and PV were less than
0.0001. Coefficients of determination (R2 and adjusted R2 values)
were used to examine the accuracy of the generated models. The R2
value was 0.9954 and the adjusted R2 was 0.9921 for the response of
FFA, while the R2 value was 0.9365 and the adjusted R2 was 0.9019
for the response of PV in neutralized kenaf seed oil. The response
surface models could explain 99.5% of the variation in the FFA and
93.65% of the variation in the PV, thus indicating that the variability
of responses was explained well by the models. Both of the mod-
els demonstrated a difference of less than 0.2 from the adjusted
R2 and predicted R2 , which indicates that these values are in rea-
sonable agreement. Lack of fit test in the ANOVA can evaluate the
adequacy of the model to fit the experimental data. The ANOVA for
the lack of fit test for all the responses were insignificant (p > 0.05)
indicates that the models were adequately fitted the experimental
data for all the response variables. Thus, well-fitting models for FFA
and PV in neutralized kenaf seed oil were successfully generated.

Please cite this article in press as: Chew, S.-C., et al., Optimization of neutralization parameters in chemical refining of kenaf seed oil by
response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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Table 2
ANOVA results for the response surface quadratic model on the FFA of neutralized kenaf seed oil.

Source Sum of squares Degree of freedom Mean square F-value p-value

Model 1.08 7 0.15 306.39 <0.0001

Residual 5.04 × 10−3 10 5.04 × 10−4
Lack of fit 3.75 × 10−3 7 5.36 × 10−4 1.25 0.4689
Pure error 1.29 × 10−3 3 4.30 × 10−4
Correction total 1.09 19

R2 = 0.9954; Adjusted R2 = 0.9921; Predicted R2 = 0.9835.

p-value < 0.05 indicates statistical significance.

Table 3
ANOVA results for the response surface quadratic model on the PV of neutralized kenaf seed oil.

Source Sum of squares Degree of freedom Mean square F-value p-value

Model 2.03 6 0.34 27.05 <0.0001

Residual 0.14 11 0.01
Lack of fit 0.10 8 0.01 0.91 0.5944
Pure error 0.04 3 0.01
Correction total 2.22 19

R2 = 0.9365; Adjusted R2 = 0.9019; Predicted R2 = 0.7685.

p-value < 0.05 indicates statistical significance.

Table 4
Regression coefficients and p-values for FFA in neutralized kenaf seed oil after back-
ward elimination.
Variablesa Regression coefficients
Intercept 1.03193
X1 −0.42310
X2 −4.74937 × 10−3
X3 −4.44819 × 10−3
X1 X2 −2.04295 × 10−3
X1 2 0.07324
X2 2 1.15006 × 10−4
X3 2 8.33442 × 10−5
a
X1 (Excess level of NaOH), X2 (Temperature) and X3 (Time).
b
p < 0.05 indicates statistical significance.
3.2. Effect of neutralization parameters on FFA
The content of FFA is used to measure the extent to which
triglycerides in oil has been decomposed by lipase and other actions
such as light and heat. Determination of FFA is often used to indi-
cate the oil quality and its suitability for edible. Offensive odor and
taste will result in the oil during the long term storage due to the
presence of FFA and other fatty materials in oil (Aluyor et al., 2009).
Thus, the removal of FFA is an improvement in the oil quality. One
of the main concerns of refining process is to remove FFA from the
crude oil. In view of this mathematical model, the significance of the
investigated factors and the interactions between the investigated
factors were examined. The responses were sufficiently explained
by the regression equation within the experiment design. Response
surface was successful established and it was feasible using these
regression models to predict the effects of various parameters on
the neutralization stage. The coefficients of the regression equation
and p values for the significant terms for FFA in neutralized kenaf
seed oil are shown in Table 4.
Removal of FFA in the neutralization stage of refining process
for kenaf seed oil was negatively affected by all three parameters.
Among the three parameters, excess level of NaOH (X1 ) and tem-
perature (X2 ) was the most strongly affected the removal of FFA,
followed by time (X3 ). The ANOVA shows that the first-order terms
of excess level of NaOH (X1 ) and temperature (X2 ), cross-product
term between excess level of NaOH and temperature (X1 X2 ), and
second-order terms of excess level of NaOH (X1 2 ) and temperature
(X2 2 ) had highly significant (p < 0.001) effects on the reduction of
FFA in neutralized kenaf seed oil. However, the first-order term of
time (X3 ), cross-product term between excess level of NaOH and
time (X1 X3 ), cross-product term between temperature and time
(X2 X3 ), and second-order term of time (X3 2 ) had no significant
p-valuesb effects (p > 0.05) on the reduction of FFA in neutralized kenaf seed
<0.0001 oil.
<0.0001 Fig. 1a shows the FFA decreased when the excess level of NaOH
<0.0001 was increased from 0 to 3.75%, while FFA increased when the excess
0.3483
level of NaOH was increased from 3.75 to 5.0%. The addition of a sto-
0.0007
<0.0001 ichiometric ratio of NaOH solution to the degummed oils based on
0.0002 the amount of FFA, and some excess percentages to ensure com-
0.0990 plete reaction and to make sure that no alkali was left over in oil
(Dumont and Narine, 2008). Normally, NaOH solution was added
at 0.1 to 0.5% of the excess level into the crude oil or degummed oil,
besides the addition of a stoichiometric amount of NaOH (Wang
and Johnson, 2001; Lee et al., 2014; Wei et al., 2015). However,
Karabulut et al. (2005) and Ravinder et al. (2015) reported that an
excess level of 25% and 10% of NaOH solution was added besides the
calculated amount of NaOH required in the neutralization of hazel-
nut oil and silkworm oil, respectively. Thus, 0 to 5% of the excess
level of NaOH solution was selected to study besides adding the
stoichiometric quantity of NaOH based on the preliminary exper-
iments. The result showed that a stoichiometric quantity of NaOH
solution with 3.75% excess was sufficient to neutralize the FFA in
this study. The neutralized oil is needed to wash with 10–20% of
water (90–95 ◦ C) to remove the residual soaps from the oil (Ghazani
and Marangoni, 2013).
Fig. 1b shows the FFA remained almost the same level while
the temperature was increased from 25 to 55 ◦ C, while FFA became
increased when the temperature was increased from 55 to 85 ◦ C.
Previous literature reported that the neutralization was normally
carried out at a temperature of 65 to 90 ◦ C (Wang and Johnson,
2001; Karabulut et al., 2005; Farhoosh et al., 2009; Lee et al., 2014).
However, the result in this study showed that a temperature of
40 to 50 ◦ C was more efficient to neutralize the FFA. The previous
study reported that neutralization was insufficient to neutralize
the FFA at 25 ◦ C and neutralization showed similar efficiency at the
temperature of 45, 65 and 85 ◦ C. Thus, 45 ◦ C was selected as the
operating temperature for the neutralization stage for tea seed oil.
Besides that, a lower temperature is better to protect the bioac-
tive compounds in the oil (Wei et al., 2015). Time was not removed
by backward elimination although it had an insignificant effect on
the FFA of neutralized kenaf seed oil, as it remains to maintain the
hierarchy of the model. Previous studies reported that the neutral-
ization was normally carried out in a time period of 20 to 30 min

Please cite this article in press as: Chew, S.-C., et al., Optimization of neutralization parameters in chemical refining of kenaf seed oil by
response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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a Table 5
Regression coefficients and p-values for PV in neutralized kenaf seed oil after back-
One Factor
ward elimination.
1.2

Variablesa Regression coefficients p-valuesb

1 Intercept 3.83350 <0.0001
X1 −0.38472 <0.0001
0.8 X2 −0.02278 0.0465
X3 −0.03371 0.0019
FFA (%)

X1 X3 0.01800 0.0001
0.6
X1 2 −0.06899 0.0005
X2 2 2.45024 × 10−4 0.0297
0.4
a
X1 (Excess level of NaOH), X2 (Temperature) and X3 (Time).
b
p < 0.05 indicates statistical significance.
0.2
2
2

0 temperature decreased from 85 to 25 ◦ C. The response surface was

generated based on the fitted second-order polynomial equation
0 1.25 2.5 3.75 5
after backward elimination:

A: Excess level of NaOH (%) Y = 1.03193 − 0.42310X 1 − 4.74937 × 10−3 X 2 − 4.44819 × 10−3
b
One Factor X 3 − 2.04295 × 10−3 X 1 X2 + 0.07324X 1 2 + 1.15006 × 10−4 X 2 2
1.2
+ 8.33442 × 10−5 X 3 2
1

0.8

3.3. Effect of neutralization parameters on PV

FFA (%)

0.6

Previous studies showed that neutralization increased the PV in

0.4 the neutralized oil (Zacchi and Eggers, 2008; Bachari-Saleh et al.,
2013). Thus, PV is another index that needs to be monitored during
0.2 2
2 neutralization stage. Decreasing of PV in the neutralization stage
of refining process for kenaf seed oil was negatively affected by
0
all three parameters, excess level of NaOH (X1 ), temperature (X2 )
25 40 55 70 85 and time (X3 ). Table 5 shows that excess level of NaOH (X1 ) was
B: Temperature (ºC) the most strongly affected the PV in neutralized kenaf seed oil, fol-
lowed by time (X3 ) and temperature (X2 ). The p values from the
c
One Factor ANOVA result revealed that the first-order term of excess level of
1.2
NaOH (X1 ), cross-product term of excess level of NaOH and time
(X1 X3 ), and second-order term of excess level of NaOH (X1 2 ) had
1 highly significant (p < 0.001) effects on the decreasing of PV in neu-
tralized kenaf seed oil. However, first-order terms of temperature
0.8 (X2 ) and time (X3 ), and second-order term of temperature (X2 2 )
showed significant effects (p < 0.05) on the PV in neutralized kenaf
FFA (%)

0.6 seed oil. Cross-product terms of excess level of NaOH with temper-
ature (X1 X2 ) and temperature with time (X2 X3 ), and second-order
0.4 term of time (X3 2 ) had no significant effect (p > 0.05) on the PV of
neutralized kenaf seed oil.
0.2 2
2 Fig. 3a shows the PV decreased when the excess level of NaOH
was increased from 1.25 to 5%. Besides to neutralize FFA, neutraliza-
0
tion is a stage to remove undesirable components in the degummed
10 20 30 40 50 oil such as residual phospholipids, pigments, and waxes that might
promote oxidative reactions in the oil (Wei et al., 2015). Complete
C: Time (min) reaction of FFA with the appropriate amount of NaOH is needed.
Absorption of hydroperoxides by the soapstock might occur dur-
Fig. 1. Main effect plot showing the effect of (a) excess level of NaOH (X1 ), (b)
temperature (X2 ) and (c) time (X3 ) on the FFA of neutralized kenaf seed oil.
ing this stage. Fig. 3b shows the PV decreased slightly when the
temperature increased from 25 to 40 ◦ C, while PV was started to
increase when the temperature was increased from 40 to 85 ◦ C.
(Suliman et al., 2013; Lee et al., 2014; Wei et al., 2015). In this study, This showed that higher temperature might produce more primary
neutralization was studied in a time varied from 10 to 50 min in oxidation products. Fig. 3c shows the time had a positive linear
order to ensure the time needed for complete reaction of FFA with effect on the PV of neutralized kenaf seed oil. Increasing of PV was
NaOH. observed when the time was increased from 10 to 50 min. Longer
Fig. 2 visualizes a 3D surface plot for FFA as a function of the duration of the neutralization stage might cause more hydroper-
excess level of NaOH and temperature (X1 X2 ), which generated oxides produced. Fig. 4 shows the interaction of excess level of
based on a medium level of time (30 min). It was observed that FFA NaOH and time (X1 X3 ) on PV of neutralized kenaf seed oil based
decreased when the excess level of NaOH was in the range of 2.5 to on the medium level of temperature (55 ◦ C). A decreasing trend
3.75%. However, FFA increased beyond this range of excess level of was observed on the PV when the excess level of NaOH increased.
NaOH. On the other hand, FFA showed a decreased trend when the On the other hand, PV decreased with the decrease of time at a

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response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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1.2

0.8
F F A (% )

0.6

0.4

0.2

85 5
70 3.75
55 2.5
40 1.25
B: Temperature (ºC) A: Excess level of NaOH (%)
25 0

Fig. 2. Response surface plot of interaction between excess level of NaOH and temperature (X1 X2 ) at middle level of time on the FFA of neutralized kenaf seed oil.

higher level of excess NaOH (5%). The response surface was gen- Table 6
Physicochemical properties and fatty acid profile of crude and neutralized kenaf
erated based on the fitted second-order polynomial equation after
seed oil.
backward elimination:
Oil quality Crude Neutralized Significance
Y = 3.83350 − 0.38472X 1 − 0.02278X 2 − 0.03371X 3 + 0.01800X 1 ◦
Refractive index at 20 C 1.4611 ± 0.0003 b
1.4629 ± 0.0005 a **

X3 − 0.06899X 1 2 + 2.45024 × 10−4 X 2 2 Specific gravity at 20 ◦ C 0.90 ± 0.01a 0.88 ± 0.01b *

Peroxide value (meq/kg) 2.90 ± 0.10a 1.57 ± 0.03b ***

Free fatty acid (%) 1.94 ± 0.02a 0.12 ± 0.01b ***

Iodine value (mg/100 g) 85.40 ± 1.11a 86.79 ± 2.51a n.s.

Fatty acid profile (%)
3.4. Determination and experimental validation of optimal Myristic (C14:0) 0.14 ± 0.00a 0.14 ± 0.00a n.s.
parameters Palmitic (C16:0) 19.41 ± 0.04a 19.15 ± 0.17a n.s.
Palmitoleic (C16:1) 1.10 ± 0.20a 1.27 ± 0.01a n.s.
Validation of optimal parameters is important to ensure that Heptadecenoic (C17:1) 0.22 ± 0.01b 0.24 ± 0.00a *

Stearic (C18:0) 3.67 ± 0.02a 3.62 ± 0.02b *

the predicted results were not biased towards the practical value
Oleic (C18:1n9) 36.37 ± 0.15a 36.17 ± 0.13a n.s.
with the objective of each response to obtain maximum desirabil- Linoleic (C18:2n6) 30.86 ± 0.10a 30.70 ± 0.04a n.s.
ity using recommended optimal parameters. The objective of this ␥-Linolenic (C18:3n6) 3.22 ± 0.12b 3.54 ± 0.05a *

procedure is to determine the levels of experimental factors which ␣-Linolenic (C18:3n3) 0.69 ± 0.02b 0.73 ± 0.01a *

Arachidic (C20:0) 0.60 ± 0.02b 0.66 ± 0.01a *

would allow obtaining a neutralized kenaf seed oil with reducing
Behenic (C22:0) 0.08 ± 0.00a 0.07 ± 0.00a n.s.
the most FFA and PV. Optimum parameters in the neutralization Lignoceric (C24:0) 0.07 ± 0.00a 0.07 ± 0.00a n.s.
stage of refining process for kenaf seed oil were generated through Nervonic (C24:1) 3.58 ± 0.02a 3.66 ± 0.07a n.s.
the RSM optimization approach by highest reduction of FFA and PV. SFA 23.97 ± 0.04a 23.70 ± 0.14b *

This set of parameters was determined to be optimum by the RSM MUFA 41.27 ± 0.06a 41.33 ± 0.05a n.s.
PUFA 34.76 ± 0.03b 34.97 ± 0.09a *
optimization procedure and balance of various aspects. The rec-
ommended optimum parameters were used to test the adequacy SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsatu-
rated fatty acids.
of the model equations for predicting the optimum response val- ab
Means ± standard deviations followed by different superscript letters within the
ues. The optimum parameters were selected by maximizing the same row are significantly different according to t-test.
desirability of the responses. The selected optimum parameters in *
Significant at p < 0.05 level.
the neutralization of kenaf seed oil were an excess level of NaOH **
Significant at p < 0.01 level.
of 3.75%, a temperature of 40 ◦ C and 20 min of duration. Triplicate
***
Significant at p < 0.001 level.
experiments were carried out based on the recommended opti-
mum parameters within the experimental range for validation of
mended optimum condition. Thus, the good correlation between
the optimized results. A mean value of 0.12 ± 0.01% was obtained
these results confirmed that the models were sufficient for pre-
for FFA, and a mean value of 1.57 ± 0.03 meq/kg was obtained for
dicting the reduction of FFA and PV in the neutralization stage of
PV under selected optimum parameters, compared with the pre-
the refining process for kenaf seed oil.
dicted values of 0.11% (FFA) and 1.58 meq/kg (PV). There were no
significant differences in experimental and predicted values for FFA
(p = 0.112) and PV (p = 0.484), as revealed by one-sample t-test. The 3.5. Oil quality and fatty acid profile
experimental values agreed with the predicted values at a level
of significance of 0.05. The closeness between the experimental Table 6 shows the physicochemical properties and fatty acid
values and predicted values confirmed the validity of the recom- profile of crude and neutralized kenaf seed oil under optimal

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response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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a One Factor
3

2.5

P V (m e q /k g )
2
2
2

1.5

0 1.25 2.5 3.75 5

A: Excess level of NaOH (%)

b One Factor
3

2.5
P V (m e q /k g )

2
2
2

1.5

25 40 55 70 85

B: Temperature (ºC)

c One Factor
3

2.5
P V (m e q /k g )

2
2
2

1.5

10 20 30 40 50

C: Time (min)

Fig. 3. Main effect plot showing the effect of (a) excess level of NaOH (X1 ), (b) temperature (X2 ) and (c) time (X3 ) on the PV of neutralized kenaf seed oil.

parameters. There was a significant increase in the refractive index tribute to the increase in refractive index in this study, which
of neutralized kenaf seed oil from the crude oil. The slight increase supported by the results of Pandurangan et al. (2014) and Chew
in unsaturated fatty acids after neutralization stage might con- et al. (2016). Removal of gums and some coloring compounds has

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response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
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2.5
P V (m e q /k g )
1.5
0.5
50
40
30
20
C: Time (min)
10
Fig. 4. Response surface plot of interaction between excess level of NaOH and time (X1 X3 ) at middle level of temperature on the PV of neutralized kenaf seed oil.
affected the weight of the oil, which resulted in the slight decrease
of specific gravity from crude oil to neutralized oil (Aluyor et al.,
2009). Neutralization stage was helped to remove hydroperoxides
content, as indicated by the significant decrease (p < 0.001) in the PV
of neutralized oil. Neutralization increased the level of PV in soy-
bean oil was showed by a previous study (Farhoosh et al., 2009).
This might due to high temperature used (85–90 ◦ C) in the neu-
tralization stage, which promotes the occurrence of the oxidative
reaction. There was a decrease of 93.8% of the FFA after the neutral-
ization stage with optimized parameters. The majority of FFA had
been removed during the neutralization stage. The previous study
was only removed 43.6% of FFA in the crude kenaf seed oil after neu-
tralization stage without optimizing the parameters (Chew et al.,
2016). The dosage of NaOH and temperature used are the main dif-
ference in the neutralization stage of kenaf seed oil in this study
and previous study. Neutralization stage in the previous study was
carried out at 65 ◦ C with an excess level of 0.2 to 0.5% of the stoichio-
metric quantity of NaOH solution, which was not efficient to reduce
FFA. There was a slight increase in the iodine value of neutralized oil
but the difference was not significant (p > 0.05). Neutralization did
not affect the degree of unsaturation of the oil significantly. Crude
kenaf seed oil consisted predominantly of monounsaturated fatty
acid (41.27%), followed by polyunsaturated fatty acid (34.76%) and
saturated fatty acid (23.97%). Oleic acid (C18:1n9) represented the
most predominant fatty acid, followed by linoleic acid (C18:2n6)
and palmitic acid (C16:0). Neutralization did not affect so much in
the fatty acid profile of kenaf seed oil in this study. Karabulut et al.
(2005) and Aluyor et al. (2009) reported that refining process did
not have a significant effect on the fatty acid profile of hazelnut oil
and groundnut oil, respectively. The total unsaturated fatty acid is
76.03% in crude oil and 76.30% in neutralized oil. The high amount
of unsaturated fatty acids makes the kenaf seed oil suitable to use
as cooking oil for diet enrichment.
4. Conclusion
Nowadays, market competition is forcing the oil producers to
optimize the refining process for different types of edible oil. Mod-
Please cite this article in press as: Chew, S.-C., et al., Optimization of neutralization parameters in chemical refining of kenaf seed oil by
response surface methodology. Ind. Crops Prod. (2016), http://dx.doi.org/10.1016/j.indcrop.2016.11.043
5
3.75
2.5
1.25
A: Excess level of NaOH (%)
0
eling of neutralization stage of the chemical refining process with
central composite design of RSM gives an insight into the effects
of the processing parameters and interactions between the param-
eters on the reduction of FFA and PV in kenaf seed oil during the
neutralization stage. The results of ANOVA show that regression
models were statistically good with a significance level of p < 0.0001
and the models had no significant (p > 0.05) lack of fit. Thus, well-
fitting models were successfully generated and represent a good
predictor of the experimental results. The optimum parameters in
the neutralization stage of refining process for kenaf seed oil were
an excess level of NaOH of 3.75%, temperature of 40 ◦ C and time
of 20 min, which reduced the FFA to 0.12% and reduced the PV to
1.57 meq/kg in neutralized kenaf seed oil. This study could be use-
ful for the development of industrial refining process of kenaf seed
oil that beneficial in food and nutraceutical industries.
Acknowledgement
Financial support of this work by internal funding from CERVIE
UCSI University (Proj-In-FAS-024) is gratefully acknowledged.
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