Journal of Food Engineering 89 (2008) 472–478

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Journal of Food Engineering

journal homepage: www.elsevier .com/locate / j foodeng

Supercritical fluid extraction of b-carotene from crude palm oil using CO2

R. Davarnejad, K.M. Kassim, A. Zainal *, Suhairi A. SataSchool of Chemical Engineering, Engineering Campus, University Science Malaysia, 14300 Nibong Tebal, Penang, Malaysia

a r t i c l e i n f o

Article history:Received 24 December 2007Received in revised form 26 February 2008Accepted 31 May 2008Available online 7 June 2008

Keywords:Supercritical extractionb-CaroteneCrude palm oilCO2 solventYield

0260-8774/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2008.05.032

* Corresponding author. Tel.: +60 45996462; fax: +E-mail address: chzahmad@eng.usm.my (A. Zainal

a b s t r a c t

The aim of this study was to ascertain the influence of pressure, temperature and time on the supercrit-ical fluid extraction of b-carotene from the crude palm oil. The operating conditions were shown as fol-lows: pressures of 75, 125 and 175 bar, temperatures of 80, 100 and 120 �C and extraction time of 1, 3 and5 h. The extracts were analyzed using UV spectroscopy at a wavelength of 450 nm. Then the experimentaldata was compared with the data obtained using a statistical method. The results from the model showeda good agreement with the experimental data. The results (obtained from the statistical model) demon-strate that a pressure of 140 bar, temperature of 102 �C and extraction time of 3.14 h are required toobtain optimum yield of b-carotene (1.028 � 10�2%) from the extraction process, however the maximumyield of the b-carotene (1.741 � 10�2%) was experimentally obtained at a pressure of 75 bar, temperatureof 120 �C and extraction time of 1 h.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Palm oil is the main export product of Malaysia, which hasabout 1.9 million ha of oil palm plantations. The main productfrom oil palm plantations is palm oil which is a crude palm oil(CPO). The crude palm oil is obtained by mechanical extraction ofpalm fruits. CPO, with melting point of 36 �C is a semi-solid mate-rial at ambient temperature (Gunstone, 1987). It has a significantamount of carotene that can be extracted. Conventional methodsbased on the solvent extraction of carotene from natural productssuch as CPO are time consuming since they require multipleextraction steps and need a large amount of organic solvents,which are often expensive and potentially harmful. On the otherhand extraction with carbon dioxide under supercritical conditionsconstitutes an emerging technology in terms of environmental im-pact. The advantages of using carbon dioxide include its non-toxicnature, chemical inertness, low cost and ready availability (Haw-thorne, 1990). Furthermore, the use of supercritical carbon dioxidehelps to minimize the thermal degradation of natural products(Macías-Sánchez et al., 2005). b-Carotene is a precursor of vitaminA in human and animal metabolism and it is used in the food pro-cessing industry for coloring purposes (Cygnarowicz et al., 1990).The main interest in the use of carotenoids is because, unlike manyother dyes, they are not affected by the presence of ascorbic acid orheating and freezing cycles. Furthermore, carotenoids are extre-mely strong dyes which impart the desired properties to foodseven at levels of parts per million. Carotenoids are increasinglyused in food technology, mainly due to consumer pressure and

ll rights reserved.

60 45941013.).

more demanding regulations regarding the use of artificial dyes(Macías-Sánchez et al., 2005).

Recently, various methods for extracting carotenes from palmoil have been developed. These methods include saponificationprocess (Nitsche et al., 1999), adsorption using selective solventextraction (Tanaka, 1986; Ooi and May, 2000) and combinationof transesterification and molecular distillation process (Ooi,1994). Chuang and Brunner transesterified triglycerides from CPOwith methanol using base catalyst. Afterwards, the glycerol wasseparated and the product was washed with water to remove thecatalyst and methanol. Then, supercritical fluid extraction usingCO2 was applied to enrich crude palm oil from caroteniods up to200-fold (Chuang and Brunner, 2006).

b-Carotene in palm oil fractions at 200 bar and 40, 50 and 60 �Cwas determined by Markom et al. (2001). It was concluded thattemperature had a little effect on the solubility of b-carotene insupercritical CO2. Furthermore, the addition of polar co-solventssuch as ethanol did not affect the extraction of carotenes fromCPO (Markom et al., 2001). Birtigh et al. (1995) showed that b-car-otene solubility increased with increasing pressure (at constanttemperature of 40 �C). Many researchers have studied on the solu-bility of b-carotene in supercritical extraction process such as Cyg-narowicz et al. (1990), Hartono et al. (2001), Macías-Sánchez et al.(2005) and Gast et al. (2001).

2. Experimental

2.1. Materials

Crude palm oil (as feed) was purchased from United Oil PalmIndustries Sdn. Bhd., Nibong Tebal, Penang, Malaysia. CO2 as solvent

Nomenclature

CPO crude palm oilP pressure (bar)T temperature (�C)t extraction time (h)Y yield of b-carotene extraction based on model

Yield g extracted b-carotene per g crude palm oil (feed)wE b-carotene weight fraction in the extracted (vapor)

phasewS b-carotene weight fraction in the liquid (solute) phase

R. Davarnejad et al. / Journal of Food Engineering 89 (2008) 472–478 473

(99.99%) was purchased from MOX Sdn. Bhd., Malaysia. b-carotene(96%, Fluka), acetone (99.8%, Baker) and n-hexane (99.8%, Baker)were used for analysis.

2.2. Chemical analysis

Samples were analyzed using a spectrophotometer (model UV-120-020, Shimadzu, Tokyo, Japan) after liquid phase extraction.They were diluted in a solvent mixture of 70% n-hexane and 30%acetone (v/v) and absorbance was read at 450 nm wavelength.Carotenes in the samples were calculated in terms of b-carotene,using a standard absorbance curve calibrated with b-carotene. A100 ml stock solution was prepared with 0.1 g of b-carotene in7:3 (v/v) solvent mixture of n-hexane and acetone. Aliquots weretaken from this solution and diluted to five different concentra-tions. Absorbance was read at 450 nm.

2.3. Laboratory procedure

A rate test supercritical extraction apparatus (fabricated by AL-PHA DYNAMIC Sdn. Bhd., Penang, Malaysia) as shown in Fig. 1 wasused in this study. The experimental set-up consisted of an extrac-tor (volume 500 cm3) equipped with a spray (1), a re-circulatingpump (two phases reciprocating pump, Haskel brand from USAand model: DSF-35 which works by using an air compressor withmaximum compressed air pressure of 150 psi) (2), two traps (3),receiver (volume 200 cm3) (4), two pressure gauges (5, 6) and ahigh pressure pump (7) as shown in Fig. 1. The extractor was in-serted into the oven and the other parts were isolated by wrappingwith glass wool.

The operating methodology involved loading the rate testextraction apparatus with approximately 200 cm3 of the CPO sam-ple. CO2 at cylinder pressure was admitted into the cell. A highpressure pump connected between the cylinder containing thecontacting CO2 and the cell was then switched on to pressurize

CO2

(1)

(4)

(3)

(2)

(6)

(5)

(7)

Fig. 1. Supercritical fluid extraction apparatus.

the cell. The pressure was adjusted by opening the trap valve. Atthis stage, the heater for the air bath (oven) and the bath fan wereswitched on and the temperature was set at required level. Whenthe desired temperature was reached, the pressure was found tohave slightly increased. The pressure was then re-adjusted byopening the trap valve. The re-circulating pump was switched onand the system was left to achieve steady state condition. The stea-dy state condition was attained when the pressure and tempera-ture were both constant. The timer was then switched on andthe system was left to run. The system was switched off at the pre-determined time during the extraction process.

2.4. Statistical analysis

Empirical correlations were obtained using the experimentaldata and the Design-Expert version 6.0.6 software. Response sur-face method (central composite) was employed to collect data bythree factors and three levels of variable combinations. Responsesurfaces can be illustrated as three-dimensional plots by repre-senting the response as a function of 2-factors and keeping theother constant. The experimental domain involved three factorsand 20 runs. The 20 experimental points consisted of 14 factorialpoints and 6 central points (the repeated points). The triplicationof center points was used to estimate the experimental error. Themeasured responses were defined as the % of yield of b-caroteneextraction. Optimization was also carried out independently.

3. Results and discussion

3.1. Experimental results

Liquid phase (in the cell) was weighed and analyzed before andafter the extraction process. Then, by applying the mass balancemethod the extracted phase data were calculated.

The experimental results involving b-carotene extraction yieldpercentage (yield%), b-carotene weight fraction percentage in thevapor (extracted) phase (wE%) and b-carotene weight fraction per-centage in the liquid (solute) phase (wS%) were listed in Tables 1–3at various pressures and at temperatures of 80, 100 and 120 �C,

Table 1Yield of carotene extraction, weight fraction in extracted phase and liquid phase atvarious pressures, at extraction times 1, 3 and 5 h; temperature = 80 �C

P (bar) Yield � 102% wE � 102% wS � 102%

1 h and 80 �C175 1.312 5.526 1.644125 0.916 19.764 1.72875.0 1.015 6.391 1.842

3 h and 80 �C175 1.205 7.278 1.471125 0.987 17.940 1.67075.0 1.099 12.478 1.608

5 h and 80 �C175 1.537 4.609 1.542125 1.353 8.147 1.45475.0 1.066 0.090 1.701

Table 2Yield of carotene extraction, weight fraction in extracted phase and liquid phase atvarious pressures, at extraction times 1, 3 and 5 h; temperature = 100 �C

P (bar) Yield � 102% wE � 102% wS � 102%

1 h and 100 �C175 1.097 3.069 1.754125 1.064 8.336 1.72275.0 0.996 18.869 1.657

3 h and 100 �C175 1.073 8.032 1.722125 1.031 6.330 1.82675.0 1.263 6.260 1.632

5 h and 100 �C175 1.232 22.916 1.712125 1.170 8.661 1.61375.0 1.301 16.523 1.416

Table 3Yield of carotene extraction, weight fraction in extracted phase and liquid phase atvarious pressures, at extraction times1, 3 and 5 h; temperature = 120 �C

P (bar) Yield � 102% wE � 102% wS � 102%

1 h and 120 �C175 1.191 4.299 1.902125 1.368 7.074 1.48475.0 1.741 6.359 1.134

3 h and 120 �C175 1.145 3.924 1.877125 1.196 5.823 1.62875.0 1.253 31.509 1.364

5 h and 120 �C175 0.927 41.224 1.676125 1.533 6.002 1.38675.0 1.534 17.402 1.130

Table 4Experimental program and results for the yield of b-carotene supercritical extractionby CO2 from CPO

Run no. P (bar) T (�C) t (h) Yield � 102%

1 75 80 1 1.0162 175 80 1 1.3123 125 100 3 1.0204 75 120 5 1.5345 175 80 5 1.5386 75 120 1 1.7417 125 100 3 1.0378 125 100 3 1.0409 175 120 1 1.19110 175 120 5 0.92711 125 100 3 1.03912 75 80 5 1.06613 125 100 5 1.17014 175 100 3 1.07315 125 120 3 1.19616 125 100 3 1.04317 75 100 3 1.26318 125 100 3 1.03519 125 100 1 1.06420 125 80 3 0.987

474 R. Davarnejad et al. / Journal of Food Engineering 89 (2008) 472–478

respectively. The experimental results showed that maximum ex-tracted b-carotene in the extracted phase (wE% = 31.509 � 10�2%)was obtained at pressure of 75 bar, at temperature of 120 �C andat extraction time of 3 h. Maximum value of b-carotene in the li-quid phase (wS % = 1.902 � 10�2 %) was obtained at pressure of175 bar, at temperature of 120 �C and at extraction time of 1 h.So, CPO was extremely enriched from b-carotene at the mentionedconditions.

Furthermore, the results clearly showed that maximum yield ofb-carotene (yield% = 1.741 � 10�2%) was achieved at minimumpressure (75 bar), at maximum temperature (120 �C) and at mini-mum extraction time (1 h).

3.2. Statistical analysis

The quadratic response curve generated from the experimentaldata using Design-Expert software is expressed as follows (Eq. (1)):

Y ¼ ð�5:31608� 10�3Þ þ ð9:68882� 10�5 PÞ þ ð1:46142

� 10�4 TÞ þ ð9:50160� 10�4 tÞ þ ð5:1111� 10�7 P2Þ

þ ð1:29193� 10�6 T2Þ þ ð1:91119� 10�4 t2Þ

� ð2:40713� 10�6 P TÞ þ ð1:4768� 10�6 P tÞ

� ð2:33403� 10�5 T tÞ ð1Þ

where Y is the yield of extracted b-carotene expressed in g b-caro-tene per g CPO (feed), T is the temperature (�C), P the pressure (bar)and t the extraction time (h). This is the most suitable based on theexperimental data; it exhibits low standard deviation (5.105 �10�4), high R2 (0.972) and low PRESS (3.127 � 10�5).

The results related to the statistical analysis (columns 1–4) andthe yield of b-carotene extraction (column 5) which was experi-

mentally determined, were listed in Table 4. Low yield is observedin b-carotene supercritical fluid extraction using CO2 solvent. Itseems to be reasonable because b-carotene is always enriched inthe liquid phase. The literatures also confirm that supercriticalCO2 has a low solubility for b-carotene most of which is concen-trated in the liquid phase (Markom et al., 2001; Ooi et al., 1996;Jungfer and Brunner, 1999).

The yield of b-carotene extraction in relation to the extractionpressure, extraction temperature and extraction time is shown inFig. 2. The extraction pressure, extraction temperature and timeof extraction were fixed at middle values of 125 bar, 100 �C and3 h as shown in Fig. 2a–c, respectively. The significant of the inves-tigated factor and their interaction were examined. It is clearlyseen that there is a high dependence of yield of b-carotene extrac-tion on pressure as the absolute value of t (ratio of difference ofsample mean to standard error of difference of sample means) isgreater than 2.5 at P-level (probability of acceptance) less than0.05 corresponding to variable pressure (Steiner, 1984). In a similarfashion, temperature was shown to be significant whereas time ofthe extraction was shown to be not significant within the rangestudied. The second order terms (P2, T2 and t2) of the three processparameters show significant effect on yield of b-carotene extrac-tion. The two-level interactions show that the interdependenceof pressure and temperature (the cross-product term PT) had moresignificant effect on the yield of b-carotene extraction. Likewise theeffect of the cross-product term Tt on yield of b-carotene extrac-tion was also significant. ANOVA of the model showed a moder-ately good F value (Fisher’s variance ratio) indicating thatinteraction among the variables is not negligible.

The optimal processing conditions (P, T and t) for the yield of b-carotene extraction from CPO were determined with a multipleregression relationship. The complete quadratic model showed avery good fit and the data were correlated well. The response sur-faces have been shown in the stereoscopic figures (Fig. 2a–c).Fig. 2a shows the yield of b-carotene extraction as related toextraction pressure and extraction temperature (extraction timewas set at 3 h). This figure showed that maximum yield of b-caro-tene extraction (1.58296 � 10�2%) was obtained at pressure of75 bar, temperature of 120 �C and extraction time of 3 h (as actualfactor). Fig. 2b shows the yield of b-carotene extraction as relatedto extraction pressure and extraction time (extraction temperaturewas set at 120 �C). This figure showed that maximum yield of b-carotene extraction (1.26480 � 10�2%) was obtained at pressureof 75 bar, extraction time of 5 h and temperature of 100 �C (as

0.95669

1.11326

1.26982

1.42639

1.58296

Yield×102

75.00

100.00

125.00

150.00

175.00

80.00

90.00

100.00

110.00

120.00

A: pressure B: temperature

1.03097

1.10404

1.17711

1.25019

1.32326

Yield×102

75.00

100.00

125.00

150.00

175.00

1.00

2.00

3.00

4.00

5.00

A: pressure C: time

a

c

b

0.99865

1.08271

1.16678

1.25084

1.33490

Yield×102

80.00

90.00

100.00

110.00

120.00

1.00

2.00

3.00

4.00

5.00

B: temperature C: time

Fig. 2. (a) Yield of b-carotene extraction as a function of extraction pressure and extraction temperature (extraction time was set at 3 h). (b) Yield of b-carotene extraction as afunction of extraction pressure and extraction time (extraction temperature was set at 120 �C). (c) Yield of b-carotene extraction as a function of extraction temperature andextraction time (extraction pressure was set at 125 bar).

R. Davarnejad et al. / Journal of Food Engineering 89 (2008) 472–478 475

actual factor). Fig. 2c shows the yield of b-carotene extraction asrelated to extraction temperature and extraction time (extractionpressure was set at 125 bar). This figure showed that maximumyield of b-carotene extraction (1.3349 � 10�2%) was obtained attemperature of 120 �C, extraction time of 1 h and pressure of125 bar (as actual factor).

Based on the statistical test used for the regression models (F-test, t-test and ANOVA) as discussed above, it is seen that the com-bination of extraction pressure and extraction temperature had themost significant effect on the yield of b-carotene compared to anyother combination of extraction parameters.

To determine the optimal processing conditions of extraction ofb-carotene from CPO, i.e., to determine the optimal values of P, Tand t, the first partial derivatives of the regression equation wascarried out with respect to P, T and t and set to zero. The point thusobtained is known as the stationary point, (P = 140 bar, T = 102 �Cand t = 3.14 h).

4. Conclusion

Supercritical fluid extraction as a clean method can be appliedfor separating or enriching vitamins and natural products. From

the experiments, the highest yield of b-carotene extraction(1.741 � 10�2%) was obtained at a pressure of 75 bar, temperatureof 120 �C and extraction time of 1 h. It is concluded that CPO is en-riched from b-carotene and increases its edible value by supercrit-ical fluid extraction process. Results obtained from the statisticalmodel were in good agreement with those from the experiment.The model predicted well the yield of b-carotene extraction inthe studied operating range. Statistical analysis indicated thatpressure and temperature significantly affect the yield of b-caro-tene extraction. The optimize point was estimated as pressure140 bar, temperature 102 �C and extraction time 3.14 h at whichthe yield was 1.028 � 10�2%.

Acknowledgments

Our real gratitude goes to Professor G. Ali Mansoori from Uni-versity of Illinois, Chicago, Dr. Ahmad Zuhairi Abdullah from Uni-versiti Sains Malaysia and Dr. Mohamed Hasnain Isa fromUniversiti Teknologi PETRONAS who helped and guided us. Weare also thankful to The Ministry of Science, Technology and Inno-vations, Malaysia for providing financial support (IRPA Grant No:6012616) for this work.

476 R. Davarnejad et al. / Journal of Food Engineering 89 (2008) 472–478

Appendix A

B.1. Calibtation curve for β-carotene.

B.2. Plot of observed vs. predicted values of response yield (% of total β -carotene extracted).

00.0020.0040.0060.008

0.010.0120.0140.0160.018

0.02

0 1 2 3 4 5 6 7 8 9 10 1112 1314 15 1617 1819 20

Experimental run numbers

Obs

erve

d &

pred

icte

d va

lues

of

Yiel

d

ObservedPredicted

y = 0.5535x - 0.005R2 = 0.9947

0

0.05

0.1

0.15

0.2

0.25

0 0.1 0.2 0.3 0.4 0.5

Carotene concentration (ppm)

Abs

orpt

ion

Appendix B

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