ORIGINAL ARTICLE

Optimization of supercritical carbon dioxide extractionparameters of cocoa butter analogy fat from mango seedkernel oil using response surface methodology

Md. Jahurul Haque Akanda & Md. Zaidul Islam Sarker &

Nik Norulaini & Sahena Ferdosh &

M. Moklesur Rahman & A. K. Mohd Omar

Revised: 31 October 2012 /Accepted: 19 March 2013# Association of Food Scientists & Technologists (India) 2013

Abstract Mango seed kernels (MSK) are discarded as ag-ricultural wastes of industrial processing’s by-product aswell as direct consumption of the mango fruits. The extrac-tion parameters of SC-CO2 were optimized using centralcomposite design (CCD) of response surface methodology(RSM) yielding cocoa butter analogy fats from MSK. Thepressure, temperature, and CO2 flow rate were considered asvariables, where the linear and quadratic effect of the flowrate of CO2, quadratic effect of pressure, and interactionbetween pressure and temperature was positive and mostsignificant on the MSK oil yield. On the other hand, lineareffect of pressure and temperature, quadratic effect of tem-perature, and interaction between temperature and CO2 flowrate had a less impact. The optimized oil yield was predictedto be 11.29 % at 44.2 MPa, 72.2 °C and CO2 flow rate of3.4 ml/min which was close to the oil yield (11.7 %) ofSoxhlet extraction method. However, the stearic (41.99 to42.21 %) and oleic (43.78 to 43.89 %) the predominant fattyacids in terms of triglycerides constituents were found inMSK oils extracted by SC-CO2 and Soxhlet methods. TheMSK oil extracted using SC-CO2 method could be regardedas premium quality cocoa butter analogy fats in this study.

Keywords Supercritical carbon dioxide (SC-CO2)extraction . Mango seed kernel oil . Cocoa butter analogyfats . Optimization . Fatty acid constituents

Introduction

Mango (Mangifera indica L.) fruit is the most importantpopular seasonal fruits which is widely cultivated aroundthe world. As it is a seasonal fruit, about 20 % of mangofruits are processed for the purpose of making puree, nectar,leather, pickles, canned slices and chutney. The popularityof these products has been increasing worldwide (Loelillet1994). After industrial processing or consumption of thefruits, significant amount of MSKs are discarded as agricul-tural wastes which have adverse effects on the environment.Moreover, food manufacturers are used to spend hugeamount for these waste treatments. On the other hand,MSK contain considerable amount of cocoa butter analogyfats. Depending on the varieties, the mango seed representsfrom 10 % to 25 % of the whole fruit weight and the kernelinside the seed represents from 45 % to 75 % of the seed andabout 20 % of the whole fruit (Arogba 1997; Hemavathy etal. 1988). MSK fats have been shown to be a good source ofedible fats. In recent years, there has been an increasinginterest in MSK fats among scientists because some of theirunique physical and chemical characteristics show resem-blance to those of cocoa butter (Ali et al. 1985; Ali andDimick 1994; Dhinigra and Kapoor 1985; Lakshminarayanaet al. 1983; Narashima-Char et al. 1977).

Cocoa butter is the most important class of specialty fatswith demonstrated health benefits, and it is used entirely formaking chocolate and other confectionery products (Kim etal. 2011; Wollgast and Anklam 2000). Moreover, it is ahighly demanded fat among tropical plant fats, but its world

M. J. H. Akanda : S. Ferdosh :A. K. M. Omar (*)School of Industrial Technology, Universiti Sains Malaysia,Minden 11800, Penang, Malaysiae-mail: akmomar@usm.my

M. Z. I. Sarker (*) :M. M. RahmanDepartment of Pharmaceutical Technology, Faculty of Pharmacy,International Islamic University, Kuantan Campus,25200, Kuantan, Pahang, Malaysiae-mail: zaidulsarker@yahoo.com

N. NorulainiSchool of Distance Education, Universiti Sains Malaysia, Minden,Penang 11800, Malaysia

J Food Sci TechnolDOI 10.1007/s13197-013-0979-x

production is decreasing by the day due to the cultivationdifficulties, weather event, low productivity, and pest attacks(Bootello et al. 2012). In recent years, cocoa price is in-creasing due to increasing consumption of chocolate prod-ucts that contain higher level of cocoa butter (Afoakwa2010). Therefore, the food industries are in need for cocoabutter substitute that might have similar quality of naturalcocoa butter to meet the demand of premium grade andrelatively cheaper cocoa butter overcoming the uncertaintyin the supply of original cocoa butter due to its lessexistibility in the cocoa beans. Thus, interest of cocoa butteranalogy fats from various natural sources are increasing.

Traditional solvent extraction (mainly n-hexane) withSoxhlet is the current method for the extraction of mangoseed kernel oils. The major disadvantage of solvent extrac-tion method is that the toxic solvent residues left in theextracts and the solvent are not edible. Recently, supercrit-ical fluid extraction has been widely used for the extractionof many plant seed oils. It offers several advantages over thesolvent extraction method including operation at lower tem-peratures thus retaining thermally labile components in theextracts and eliminating the toxic solvent residue in the finalproducts. Carbon dioxide used as a solvent in supercriticalextraction has gained considerable interest among foodmanufacturers. It is an ideal solvent for the extraction ofnatural oils and other bioactive components as it is non-toxic, non-explosive, relatively low cost and easy to removefrom the final products. However, to the best of the authors’knowledge, the SC-CO2 extraction of MSK oils and theoptimum operation conditions of oil yields have not beenstudied yet.

It has been well acknowledged that response surfacemethodology (RSM) is the most effective and powerfulmathematical and statistical techniques among all othermultivariate statistical techniques used both for optimizationthe extraction process and determination the effects of pro-cess parameters as well as their interactions on the outputvariables (Wang et al. 2012). RSM is able to evaluate theeffects of multiple parameters, alone or in combination onresponse variables and also predicts their behaviour undergiven set of conditions (Nagesha et al. 2004). Another majoradvantage of RSM is that it reduces the number of experi-mental trails with high efficiency (Granato et al. 2010; Shaoet al. 2008). Meanwhile, A number of researches has beenconducted using RSM to model and optimize supercriticalcarbon dioxide (SC-CO2) extraction of oils from camelhump (Shekarchizadeh and Kadivar 2012), cottonseed oil(Bhattacharjee et al. 2007), cyperus rotundus (Wang et al.2012), hemp seed oil (Porto et al. 2012), passiflora seed oil(Zahedi and Azarpour 2011; Liu et al. 2009a), pomegranateseed oil (Liu et al. 2009b), rosehip seed oil (Machmudah etal. 2007), silkworm pupal oil (wei et al. 2009) and Vetiveriazizanioides oil (Danh et al. 2009).

The main objectives of this study were to optimize theparameters namely: pressure, temperature and CO2 flow rateusing RSM for SC-CO2 extraction of mango seed kernel oilfrom MSK to achieve the highest yield. The triglycerides interms of fatty acid constituents of the MSK oil wereanalysed to compare the yields between SC-CO2 andSoxhlet extraction methods.

Materials and methods

Materials and chemicals

The mango fruit (Apple mango) were obtained from PulauPenang, Malaysia. Commercial liquefied carbon dioxide(purity, 99.9 %) was purchased from Malaysian OxygenLtd., Penang, Malaysia. The solvent n-hexane, AR Grade(Merck, Germany) was purchased from Penang, Malaysia.

Sample preparation

The mango seed kernels were separated manually andwashed with water. The MSKs were stored at −20 °C, andthen freeze dried (Model: LABCONCO, USA) at a dryingtemperature of −47 °C and under a vacuum of 0.05 bar. Thedried samples were ground into powder, sieved by certifiedtest sieves (USA standard testing sieve, A.S.T.M.E-11, W.STyler, USA) (particle size <200 μm) and then kept in des-iccators until further use for experimental analysis.

Determination of moisture content

The determination of moisture content of the ground MSKspowder were carried out using the PORIM test method no.p5.2 (1995). The moisture content of MSK powder wasfound to 4.67 %.

Soxhlet extraction

To obtained total oil yield, the soxhlet extraction was carriedout for comparison with supercritical fluid extraction. Thesoxhlet extraction was continuously performed in triplicatefor about 10 g of MSKs powder with AR grade n-hexane(250 ml) as a solvent for 8 h. After extraction, the solventwas evaporated from the extracted oil using rotary vacuumevaporator (Buchi RE 121, Germany) and then dried in anoven at 45 °C for 1 h, weighted and kept at −20 °C ready forGC analysis.

Supercritical carbon dioxide extraction

The instrumental set-up of SC-CO2 extraction process con-sists of a SC-CO2 extractor (ISCO, Inc., Lincoln, NE; Model

J Food Sci Technol

SFX 220; two extraction vessels), a high pressure syringepump (ISCO, Inc.; Model 100 DX) with a maximum capac-ity of 69 MPa, a chiller (B/L-730, YIH DER, Taipei) forCO2 liquefaction and a carbon dioxide cylinder. The volumeof each extraction cell was 2.5 ml. The SC-CO2 extractorequipped with a heated capillary restrictor (ISCO, Inc.) withan outer diameter of 50 μm and a maximum operatingtemperature of 150º C. The software (ISCO, Inc.; ModelSFX 200) which is integrated into the SC-CO2 extractorsystem, was used to control the pressure and temperature.Four gram (4 g) of ground mango seed kernel samples wereloaded into two extraction vessel and was then placed in theheating extractor unit to equilibrate the operating tempera-ture. When pressurisation was initiated, the carbon dioxidefrom the cylinder was passed through the chillier at 0º C andpumped into the heated extraction vessel by a high-pressurepump. The oil was collected from oil-rich CO2 using anexpansion valve and a heated capillary restrictor. As per theexperimental design, the oil yield was weighed using ana-lytical balance in certain time interval. The total oil yieldswere defined in percent on 100 g of mango seed kernelsample on dry basis as described in the following equation:

Oil yield %ð Þ ¼ mass of extracted oil

mass of mango seed powder� 100 ð1Þ

A flow meter integrated in the SC-CO2 extractor wasused to measure the volume of CO2 consumption after eachextraction.

Experimental design

The SC-CO2 extraction parameters were optimized using acentral composite design of RSM to obtain a highest yield.The independent parameters studied to optimize the MSKoil yield (Y) and their concentration ranges were pressure(X1) from 25 to 50 MPa, temperature (X2) from 40 to 80 °Cand CO2 flow rate (X3) from 1 to 4 ml/min. Table 1 showsthe coded and uncoded independent varaibles used in theRSM design. The concentrations of independent variableswere based on preliminary experimental results. In the pres-ent study, the experimental design was based on a centralcomposite design (CCD) consisting of three parameters,required 20 experiments with 8 (23) factorial points, 6 axial

points, and 6 replication at the central points which is shownin Table 2. All experiments were performed in randomizedorder to minimize the effect of unexplained variability in theobserved responses due to the extraneous factors (Liu et al.2009a). A second-order polynomial regression equation wasused for predicting the response variable (Y) as follows:

Y ¼ b0 þ b1X1 þ b2X2 þ b3X3 þ b11X21 þ b22X

22

þ b33X23 þ b12X1X2 þ b13X1X3 þ b23X2X3 ð2Þ

Where ‘Y’ is the response variable (yield of MSK oil, %).βo is the constant; β1, β2, and β3 are the linear; β11, β22,and β33 are quadratic, and β12, β13, and β23 are interactioneffects, respectively. X1 and Xj are the independent vari-ables. The Minitab software (version 16) was used for theoptimization, analysis of experimental data as well as crea-tion of design matrix.

Analysis of MSK oil components

The total MSK oil yields extracted by both SC-CO2 andSoxhlet extraction methods were analyzed to determine thefatty acid constituents using a gas chromatography with flameionization detector (GC-17A, Shimadzu, Osaka, Japan). Theextracted oils were melted completely and homogenized be-fore the test sample was taken. According to the PORIM TestMethods No. p3.4 (1995), the FAMEs were prepared fromextracted oil prior to GC injection. Approximately 50 mg ofthe test sample was measured into a 2 ml vial and then 0.95 mlof n-hexane was added and the mixture shaken to dissolve theoil. About 0.05 ml of sodiummethoxide was added to the vialand shaken vigorously for 5–8 s with the help of vortexmixture (Janke & Kunkel, VF2, Germany). The mixture, firstgone clear and turbid as sodium glyceroxide was precipitated.After a few minutes, 1 μl clear upper layer of the fatty acidmethyl ester was taken and injected into GC for fatty acidanalysis using PORIM Test Methods No. p3.5 (1995). TheBPX5 phase (non-polar) GC column (30 m in length, with a0.25 μm film coating, 0.25 mm ID) was used to determine thefatty acid constituents. The column temperature was set at140º C, held for 3 min, increased with a heating rate of 5ºC/min up to the final temperature of 190º C. The injector anddetector temperatures were retained at 240 and 270º C respec-tively. Helium was used as carrier gas at a flow rate of2 ml/min. The resultant FAME chromatograms were identi-fied based on the elution order of the reference standardSupelco 37 component FAME mixtures (Sigma-Aldrich,Supelco, Bellefonte, Pa., U.S.A.). The mass of the FAMEswere determined using PORIM Test Methods No p3.5 (1995).It was based on the percentage represented by the area of thecorresponding peak relative to the sum of the areas of all thepeaks. To get better accuracy, a correction factor Ki Eq. (3)was used to convert the percentage of peak area into weight

Table 1 Levels and ranges of independent variables used in the RSMdesign

Independent variables Symbols Variable levels

−1 0 +1

Pressure (MPa) X1 20 35 50

Temperature (º C) X2 40 60 80

CO2 flow rate (mL/min) X3 1 2.5 4

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percentage of the FAME components Eq. (4). A correctionfactors was determined with the help of a chromatogramderived from the analysis of the FAME reference standardunder operating conditions identical with those used for thesample.

Correction factor Ki ¼ mi �P

Ai

Ai �P

mið3Þ

Weight percentage of the component ¼ Ki � Ai � 100P

Ki � Aið Þð4Þ

Where ki is the correction factor of component i, mi is theweight percentage of component i in the FAME standardsolution and Ai is the area under the peak corresponding tocomponent i.

Results and discussion

Fitting the model

The experimental and predicted total MSK oil yieldsobtained in applying 20 sets of experiments are listed in

Table 2. According to run order of RSM design, the highestyield was obtained at 6 followed by 18, 5, 15, 16, 14, 7, 13,20, 8, 9 and others (Table 2). For a better goodness of fitting,ANOVA was used to analyze the experimental data ofduplicates where the value of p less than 0.05 (p<0.05)was considered as statistically significant. The accuracyand the general availability of the model were determinedusing the values of lack-of-fit and determination of coeffi-cient (R2) (Table 3) where the determination of coefficient(R2) was 98.55 % which adequately represents the fit withthe experimental results. ANOVA also revealed that the mod-el for MSK oil yield was statistically significant (p<0.05).Paired T-test was used to compare the experimental and pre-dicted MSK oil yields. The T-test of mean difference clarifiedthe level of significance. Moreover, the predicted MSK oilyields were obtained from the regression model using the dataof the experimental oil yields and compared the experimentaland predicted yield each other where the predicted oil yieldscould be seen closer to those of experimental oil yields(Table 2). Thus, it could be concluded that the model wassuccessfully generated and represents the actual relationshipamong the variables.

The experimental oil yields were analyzed by multipleregression equation to determine the constant and coeffi-cients of the variables for second order polynomial modelwhere the linear, quadratic, and interaction effects of the

Table 2 Experimental and pre-dicted mango seed kernel oilyield (%) of central compositedesign using SC-CO2

Runnumber

Independent variables Oil yield (%)

Pressure,X1 (MPa)

TemperatureX2 (°C)

Flow rateX3 (ml/min)

Experimental (%) Predicted (%)

1 20 60 2.5 3.05 3.40

2 35 60 1 4.19 4.68

3 35 40 2.5 6.28 6.72

4 35 80 2.5 8.22 7.80

5 35 60 4 10.25 9.78

6 50 60 2.5 11.13 10.80

7 35 60 2.5 9.11 9.02

8 35 60 2.5 9.0 9.02

9 35 60 2.5 9.09 9.12

10 25.8 47.7 1.6 3.41 2.98

11 25.8 72.2 1.6 3.21 2.95

12 25.8 72.2 3.4 5.34 5.65

13 35 60 2.5 9.10 9.12

14 35 60 2.5 9.14 9.12

15 44.2 47.8 3.4 9.71 9.95

16 44.2 72.3 1.6 9.53 9.73

17 25.8 47.7 3.4 7.89 7.67

18 44.2 72.2 3.4 10.87 11.29

19 44.2 47.7 1.6 6.73 6.40

20 35 60 2.5 9.10 9.12

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variables were correlated. Table 3 shows the estimated con-stant, regression coefficients, corresponding t and p-values,and analysis of variance for the final regression model ofextraction of MSK (mango seed kernel) oil yields. Thefitting of the coefficients and the constant value to the Eq.(2) is expressed as follows:

Y ¼ 0:4798X1 þ 0:4033X2 þ 9:5154X3 � 0:0085X21

� 0:0044X22 � 0:7935X2

3 þ 0:0075X1X2

�0:0339X1X3 � 0:0443X2X3 � 30:5628

ð5Þ

Where, Y represents the experimental oil yield (%), X1,X2 and X3 represent pressure, temperature and CO2 flowrate, respectively. Applying the values of Table 3 in theequation Eq. (5), it could be seen that all the linear andquadratic effect of pressure (X1), temperature (X2) and CO2

flow rate (X3) and their interaction between pressure andtemperature (X1X2), and temperature and CO2 flow rate(X2X3) exhibited significantly (p<0.005) positive effect onthe MSK oil yields. On the other hand, the linear effect ofCO2 flow rate (X3) had positive and highly significant effecton the oil yield followed by interaction effect of pressureand temperature(X1X2), and quadratic effect of pressure(X2

1). On the contrary, the interaction between the pressureand flow rate (X1X3) had negative effect (p>0.005) on theoil yields. Based on the above model, the highest oil yieldwas achieved up to 11.29 % at optimized condition of44.2 MPa, 72.2 °C and 3.4 ml/min.

Optimization and effect of SC-CO2 extraction parameterson the yield

The response surface of MSK oil yields as a result ofindependent variables (pressure, temperature and flow rate)within the ranges were obtained using the second order

polynomial equation (Eq. 5). From the response surfaceequation, it is clear that oil yield increased with pressure,temperature and CO2 flow rate. Figure 1a shows the MSKoil yield as a function of pressure and temperature at con-stant CO2 flow rate of 2.5 ml/min. The pressure had greatlypositive effect on MSK oil yield due to the increase ofsolvent power of supercritical CO2 resulting from the incre-ment of density of CO2 (Danh et al. 2009). The oil yieldincreased with pressure and also became larger with in-creased temperature. For instance, as pressure increasedfrom 25.8 to 44.2 MPa, the oil yield increased around twofold, ie, from 3.41 % to 6.73 % at 47.7 °C and from 5.34 %to 10.87 % at 72.3 °C. These increments of oil yield can beexplained by the combined interaction phenomena betweenpressure and temperature. Danh et al. (2009) reported asimilar trend in optimization of SC-CO2 extraction of essen-tial oil from roots of Vetiveria zizanioides using RSM.

In general, at low pressures the yield decreases withhigher temperature which can be explained by the reduceddensity of supercritical CO2 at higher temperature.However, at higher pressure the yield increases with tem-peratures. In this study, the total oil yield decreased from3.41 % to 3.21 % when temperature increased from 47.7 to72.2 °C at 25.8 MPa, while at 44.2 MPa the yield increasedfrom 6.73 to 9.53 % at the same increment in temperature.The solvent density was the predominant factor at this pointto increase oil yield at pressure 44.2 MPa with increasingtemperature up to 72.2 °C. Depending on either the solutevapor pressure or the solvent density, the solubility of thesolute either increased or remained constant, or decreasedwith increase in the temperature at constant pressure (Thanaet al. 2008).

The effect of pressure and CO2 flow rate on MSK oilyields at constant temperature of 60 °C is presented in Fig.1b. Again, from the Fig. 1b, it can be seen that pressure andthe CO2 flow rate both exhibited significantly positive effecton the total MSK oil yield. It was also observed that the oilyield increased almost three times higher when the CO2

flow rate and pressure increased. For example, the yieldincreased from 4.19 to 11.13 % when the CO2 flow rateincreased from 1 to 2.5 ml/min and pressure from 35 to50 MPa, even at the constant pressure of 35 MPa, the yieldincreased from 4.19 to 10.25 % when the CO2 flow rateincreased from 1 to 4 ml/min. These increment trends of theoil yield with the CO2 flow rate and pressure are caused bythe increased solubility of MSK oil in SC-CO2 with increas-ing pressure and decreasing mass transfer resistance withincreasing CO2 flow rate. A similar trend was found for theSC-CO2 extraction and optimization of pomegranate seedoil and hazelnut oil using response surface methodology(Liu et al. 2009b; Özkal et al. 2005).

Finally, the MSK oil yield as a function of temperatureand the CO2 flow rate at constant pressure of 35 MPa is

Table 3 Estimated regression coefficients, corresponding t andp-values and analysis of variance for the final regression model ofmango seed kernel oil yield

Term Coefficients Standard error t p-value

βo −30.5628 5.53515 −5.522 0.000

X1 (β1) 0.4798 0.14349 3.344 0.009

X2 (β2) 0.4033 0.11793 3.420 0.008

X3 (β3) 9.5154 1.32240 7.196 0.000

X12 (β11) −0.0085 0.00148 −5.739 0.000

X22 (β22) −0.0044 0.00083 −5.260 0.001

X32 (β33) −0.7935 0.14833 −5.350 0.000

X1*X2 (β12) 0.0075 0.00143 5.216 0.001

X1*X3 (β13) −0.0339 0.01906 −1.780 0.109

X2*X3 (β23) −0.0443 0.01429 −3.102 0.013

R2 98.55

Adj. R2 96.95

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illustrated in Fig. 1c. As the flow rate was increased from 1to 2.5 ml/min, there was a sharp increase of MSK oil yieldfrom 4.19 to 9.14 % at a constant temperature of 60 °C.Furthermore, at a constant flow rate of 2.5 ml/min, the oilyield also increased from 6.28 to 8.22 % as temperatureincreased from 40 to 80 °C. It is evident that the oil yieldincreased as the CO2 flow rate and temperature increased,because increasing temperature enhanced the mass transferrate.

Fatty acid compositions of MSK oil

The fatty acid composition of MSK oil, in particular,palmitic, stearic, oleic and linoleic acids are comparable to

80

0 60

4

8

20

12

30 4040

50

Oil yield (%)

Temperature (°C)

Pressure (MPa)

(A)

4

30

4

2

8

20

12

30 140

50

Oil yield (%)

Flow rate (ml/min)

Pressure (MPa)

(B)

4

30

5

240

10

60 180

Oi lyield (%)

Flow rate (ml/min)

Temperature (°C)

(C)

Fig 1 Generated responsesurface plot showing the effectsof pressure, temperature andCO2 flow rate on the oil yield ata fixed (a) CO2 flow rate of2.5 ml/min, (b) temperature of60 °C and (c) pressure of35 MPa

Table 4 Fatty acid compositions (%) of MSK oil extracted by bothSC-CO2 and Soxhlet extraction methods

Fatty acid SC-CO2 (44.2 MPa,72.2 °C, 3.4 ml/min)

Hexane

Myristic acid (C14:0) 0.51±0.11 0.45±0.08

Palmitic acid (C16:0) 6.72±0.4 6.25±0.33

Stearic acid (C18:0) 41.99±1.39 42.21±1.76

Oleic acid (C18:1) 43.78±2.06 43.89±2.23

Lenoleic acid (C18:2) 4.89±0.31 4.92±0.3

Linolenic acid (C18:3) 0.83±0.18 0.91±0.2

Arachidic acid (C20:0) 1.28±0.21 1.37±0.31

Values represent average of triplicates analysis ± SD

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the fatty acid composition of cocoa butter. Those fatty acidconstituents are rarely found in plant kingdom. Recently,Muchiri et al. (2012) reported that the physico-chemicalcharacteristics of MSK oil such as iodine value (IV), sapon-ification value (SV), acid value (AV), ester value (EV),unsaponifiable matter, peroxide value, melting point, refrac-tive index, and specific gravity are similar to that of cocoabutter. The authors also suggested that the MSK oil could beused in the food industry as a substitute for cocoa butter.Table 4 shows the fatty acid compositions of MSK oilextracted by SC-CO2 at 44.2 MPa, 72.2 °C and CO2 flowrate at 3.4 and by Soxhlet extraction method using n-hexane.No significant differences were found between the oilsextracted by SC-CO2 and Soxhlet extraction methods. Thehexane and CO2 may exhibit similar behaviour forextracting oil from mango seed kernel since both of themare non-polar solvents. Similar results were observed for theextraction of Moringa oleifera kernel oils (Nguyen et al.2011), vetiveria zizanioides essential oil (Danh et al. 2009),walnut oil (Oliveira et al. 2002) and pomegranate seed oil(Liu et al. 2009b) which are in line with our observations.

The result of our study revealed that palmitic, stearic,oleic, and linoleic acids are the major fatty acids whichcomprise about 97.27 to 97.38 % of total MSK oil(Table 4). Among the fatty acid constituents, the stearic(saturated) and the oleic (unsaturated) acids were the pre-dominant fatty acids of MSK oil which accounted more than85 % of the total fatty acid contents. Abdalla et al. (2007),Ali et al. (1985) and Solís-Fuentes and Durán-de-Bazúa(2004) studied lipid classes in terms of fatty acid constitu-ents of mango seed kernel oil. These authors found that themajor fatty acids of MSK were the steric (38.2–40.2 %) andoleic acid (40.81–46.1 %) that constituted about 79.01 to86.3 % of the total fatty acids. These figures are in goodagreement with the findings of this study.

Conclusions

Optimization of SC-CO2 extraction parameters namely pres-sure, temperature and CO2 flow rate were successfully appliedusing central composite design (CCD) of response surfacemethodology (RSM) maximizing the extract of MSK oil yieldfrom MSK. The general availability of the model was deter-mined using coefficient of determination (R2) where the opti-mal condition for MSK oil yield was found to be at 44.2 MPa,72.2 °C and CO2 flow rate at 3.4 ml/min. At this condition, theMSK oil yield was predicted to be 11.29%which was close tothe yield of Soxhlet extraction (11.7 %) method. Furthermore,stearic and oleic acids were the predominant fatty acids whichaccounted for more than 85% of the total fatty acids present inthe MSK oil. Extracts with high content of such fatty acids areideal for use in formulations for CBR blends in food industry,

in particular, chocolate industry, and also extracts should beimproved specifically chemical residues free to apply in thefood and nutraceuticals industry. Thus, the MSK oil obtainedby SC-CO2 could be regarded as cocoa butter analogy fatswhich need to be further studied for physico-chemical andmorphological properties.

Acknowledgments The authors are glad to acknowledge to theUSM-Fellowship of Universiti Sains Malaysia for providing financialsupport to conduct the study.

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