Accepted Manuscript

Enrichment of β-carotene from palm oil using supercritical carbon dioxide

pretreatment-solvent extraction technique

Iftikhar, Huijun Tan, Yaping Zhao

PII: S0023-6438(17)30341-9
DOI: 10.1016/j.lwt.2017.05.026
Reference: YFSTL 6242

To appear in: LWT - Food Science and Technology

Received Date: 22 January 2017

Revised Date: 15 May 2017
Accepted Date: 16 May 2017

Please cite this article as: Iftikhar, , Tan, H., Zhao, Y., Enrichment of β-carotene from palm oil using
supercritical carbon dioxide pretreatment-solvent extraction technique, LWT - Food Science and
Technology (2017), doi: 10.1016/j.lwt.2017.05.026.

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 ACCEPTED MANUSCRIPT

1 Enrichment of β-carotene from palm oil using supercritical carbon dioxide

2 pretreatment-solvent extraction technique

3 Iftikhar, Huijun Tan, Yaping Zhao*

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4 School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,

5 800 Dong Chuan Road, Shanghai 200240, P.R China

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6 To whom correspondence should be addressed. E-mail: ypzhao@sjtu.edu.cn

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22 Enrichment of β-carotene from palm oil using supercritical carbon dioxide

23 pretreatment-solvent extraction technique

24 Iftikhar, Huijun Tan, Yaping Zhao*

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25 School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University,

26 800 Dong Chuan Road, Shanghai 200240, P.R China

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27 To whom correspondence should be addressed. E-mail: ypzhao@sjtu.edu.cn

Abstract

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29 Enriching natural 10 g/100g β-carotene concentrate further is both meaningful and challenging

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30 issue. In this paper, a two-step technique has been reported to boost the concentration of natural
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31 β-carotene. Supercritical carbon dioxide (SC-CO2) was utilized to preconcentrate 10 g/100g β-

32 carotene made from crude palm oil followed with a solvent extraction. In the first step, the β-
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33 carotene concentration was preconcentrated to 16.7 g/100g from 10 g/100g when pretreating
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34 conditions were as follows: extraction temperature 60 °C, extraction pressure 20 MPa, extraction

time 3 h, and CO2 flow rate 22 kg/h, In the second step, the β-carotene was boosted to 58.7
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36 g/100g from 16.7 g/100g when the ratio of the preconcentrated sample and n-hexane solvent was
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37 4:200 (g:mL), and the processing temperature was -5 °C. This is attributed to that SC-CO2 was

38 able selectively to remove the impurities such as oil fraction. Removing impurities enable further
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39 purification of the beta-carotene with hexane extraction. The pretreatment of removing oil
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40 fraction using supercritical (SC-CO2) plays a vital role in the enrichment of β-carotene from 10

41 g/100g β-carotene concentrates to higher content.

42 Keywords: Supercritical CO2, solvent extraction, β-carotene concentrate

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44 1. Introduction

45 Natural β-carotene is a type of pigment mostly found in vegetables, plants leaves, and fruits.

46 Dietary β-carotene can reduce the risk of lung cancer, eye and heart diseases (Holick, Michaud,

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47 Stolzenberg-Solomon, Mayne, Pietinen, Taylor, et al., 2002). β-carotene may have added

48 benefits due to its ability to be converted into vitamin A and has been widely applied in food

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49 processes and functional food (Johnson, 2002). Crude palm oil is the primary source of natural β-

50 carotene, from which many methods have been reported to extract β-carotene, such as C18

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51 reverse phase column chromatography (Choo, Yap, Ooi, Ong, & Goh, 1992), saponification

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52 (Eckey, 1949), adsorption (Khoo, Morsingh, & Liew, 1979), selective solvent extraction
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53 (Heidlas, Huber, Cully, & Kohlrausch, 1998), transesterification followed by distillation (Ooi,

54 Choo, Yap, Basiron, & Ong, 1994; Tan & Saleh, 1992), batch adsorption using a synthetic
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55 polymer adsorbent followed by solvent extraction (Latip, Baharin, Man, & Rahman, 2001),

56 supercritical fluid extraction method (Baysal, Ersus, & Starmans, 2000; Puah, Choo, Ma, &
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57 Chuah, 2005). However, these methods either could not get high content β-carotene or could not
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58 be utilized in commercial applications because of the complicated and cost process. The method

59 of combination of transesterifying crude palm oil and molecular distillation is an effective

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60 process to obtain β-carotene concentrate at present, by which the concentrate of β-carotene

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61 reached 8 g/100g (Ooi, Choo, Yap, Basiron, & Ong, 1994). However, it is tough difficult to
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62 concentrate it further. To our best knowledge, few papers on concentrating β-carotene up to more

63 than 10 g/100g regarding potential practical application were reported. Therefore, it has been a

64 great need to develop a cost-effective and scalable process to prepare high-content β-carotene.

65 The purpose of this work was to develop a scalable approach of enriching the content of 10

66 g/100g natural β-carotene concentrates made from palm oil to a desired high content by

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67 investigating the influence on the beta-carotene content of n-hexane solvent extraction,

68 supercritical CO2 extraction and the combination of supercritical CO2 and n-hexane solvent

69 extraction.

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70 2 Materials and methods

71 2.1 Materials and Chemicals

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72 The 10 g/100g β-carotene concentrate, produced from crude palm oil by a combination of

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73 transesterification and molecular distillation, was provided from Excel Vite Sdn.Bhd. CO2 gas

74 with a purity of 99.99 g/100g and Nitrogen with 99.99 g/100g were purchased from SJTU

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chemical store (Shanghai China). N-hexane and Cyclohexane with analytical grade were
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76 obtained from Ling Fing Chemical Reagent Co, Ltd.
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77 2.2 Methods

78 The general enriching technologic route is as shown in Figure 1.

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79 2.2.1 Process of enrichment of β-carotene using n-hexane extraction

80 The enrichment procedure using n-hexane extraction is provided in Figure.1. In the particular
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81 extraction process, 4 g sample was weighed and dissolved in a particular volume of n-hexane

82 solvent with magnetic stirring at 30℃ for each trial. The obtained homogeneous solution mixture
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was then settled for 12 h at -30, -15, -10, -5 and 0 ℃. After that, the solution was centrifuged at
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84 860 xg for 10 min resulting in two fractions in the centrifuge tube: the paste or solid part of the

85 bottom and the solution part of the top, which were collected, separately. The solvent in the

86 solution portion was firstly evaporated by a rotary evaporator at 50 ℃ and then was flushed out

87 by nitrogen gas. The hexane-removed residue was named as an extracted sample. The solvent in

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88 the paste or solid fraction was blown out by nitrogen gas, and called as an undissolved sample.

89 The β-carotene content of the extracted sample and the undissolved sample was analyzed,

90 respectively, by an UV-Spectrophotometer as shown in Section 2.4 below. The samples have two

91 types: one is 10 g/100g beta-carotene concentrate, and the other is 16.5 g/100g beta-carotene

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92 which is prepared by using supercritical CO2 extraction described in Section 3.2. The extraction

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93 process is same for both two types of the samples.

94 2.2.2 Pretreatment process of beta-carotene using supercritical CO2

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95 Supercritical CO2 (SC-CO2) pretreatment experiments were carried out in the supercritical CO2

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96 extraction equipment manufactured by Nantong Wise Supercritical Fluid Science and
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97 Technology Co. Ltd. As shown in Figure 2, the apparatus mainly consists of a CO2 cylinder,

98 condenser, CO2 pump, heat exchanger, extractor, and separators. The operation of each trial is
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99 briefly introduced as follows: approximately 80 g of starting materials was loaded into the
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100 basket first, equipped with both sides by steel mesh filters to avoid CO2 from carrying the solid

101 out. The basket was placed inside the extractor, and then the cap of the extractor was closed.
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102 After that, CO2 was pumped into the extractor via the condenser from CO2 cylinder, and into the
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103 separators. When the pressure and temperature of the extractor and separators reached the

104 desired preset value, CO2 started to cycle with the desired preset flow rate of 22 kg/h. The
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105 extracted sample carried out by SC-CO2 from the extractor was deposited in the separators at the
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106 pressure of around 5 MPa. The CO2 was recycled after the extracted sample settled out in the

107 separators from which the obtained sample was harvested. When the extraction was completed,

108 the residue in the extractor was gathered in a dark color glass bottle which was wrapped in an

109 aluminum foil and stored in a fridge, named as the pretreated sample for later analysis and

110 enrichment further via solvent extraction.

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111 2.2.3 Analysis of β-carotene content

112 The β-carotene content was analyzed using a spectrophotometer (model UV756PC). Weigh

113 accurately a certain amount of the sample into a 50 ml volumetric flask. Add in a few ml of

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114 cyclohexane solvent to dissolve the sample and make up to volume with the same solvent to get

115 the Stock Solution. Pipette 0.5 ml of the Stock Solution into a 50 ml volumetric flask and make

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116 up to volume with the same solvent to get a dilution sample. The resultant diluted solution was

117 transferred into1-cm transparent quartz cuvette and was scanned on the spectrophotometer in the

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118 range of 350-550 nm. The maximum absorbance of the solution was recorded. The concentration

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119 of β-carotene was calculated according to the below formula.
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 × 50 × 100


(%) =
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121 3. Results and discussion

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122 3.1 Concentrating β-carotene from its 10 g/100g concentrates using n-hexane extraction

123 We investigated the concentrating β-carotene from its 10 g/100g concentrates using an n-hexane
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124 solvent extraction. We examined the influence of temperature and the ratio of the solvent and the
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125 starting materials (mL:g). As shown in Table 1, when the solvent amount was fixed, even though
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126 the temperature decreased from -10 ℃ to -30℃, the β-carotene content varied between 11-12 %.

127 Increasing the amount of the solvent at the fixed temperature increased the β-carotene content

128 from 10 g/100g to g/100g. This may be attributed to the existence of oil components (triglyceride,

129 diglyceride, monoglyceride and fatty acid methyl ester) in the raw material, which significantly

130 prevents β-carotene separation from the solution mixture. The apparent viscosity of the mixture

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131 increased much more when the temperature was -30 ℃ even though the ratio(mL:g) between the

132 n-hexane and the starting material was 50 times. It mainly interfered with the β-carotene to be

133 settled out from the solution mixture though the solubility of β-carotene might be lower at the

temperature of -30 ℃. Therefore, it is not practicable to enrich β-carotene from 10 g/100g β-

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135 carotene concentrates via using an n-hexane extraction.

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136 3.2 Pretreating 10 g/100g beta-carotene using supercritical CO2 extraction

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137 As mentioned in Section 3.1, it was difficult to directly enrich β-carotene using a solvent

138 crystallization process because of the high content of impurity in the raw material, such as oil

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and waxes (Zaidul, Norulaini, Omar, & Smith, 2007). However, in the preliminary experiments,
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140 we found that 10 g/100g β-carotene could be concentrated further by n-hexane solvent extraction

141 when it was pretreated using SC-CO2. Therefore, we proposed a two-step approach to enrich β-
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142 carotene: 10 g/100g β-carotene was first pretreated by SC-CO2 and then the pretreated sample
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143 was further purified using n-hexane solvent extraction. In this section, we investigated the
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144 influence of process variables on the β-carotene content and the yield. The pretreatment

145 conditions and the results are listed in Table 2. Three extraction pressures, two extraction
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146 temperatures and extraction times were selected as the examining process parameters while the

147 loading amount of starting materials was fixed as 80g and CO2 flow rate as 22kg/h.
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148 The purpose of the pretreatment was to remove the impurity as much as possible and to increase
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149 β-carotene content simultaneously. The fundamental principle to remove the impurity

150 components from the 10 g/100g β-carotene concentrate is based on their solubility difference in

151 SC-CO2. Though the impurity components were not identified clearly, they mainly consisted of

152 triglyceride, diglyceride, monoglyceride, fatty acid methyl esters and wax. The solubility of these

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153 components in SC-CO2 is greater than β-carotene. Then, we can make full use of their solubility

154 difference to separate them with the β-carotene. Table 2 lists the pretreatment results from which

155 we can see that the β-carotene of all the pretreated samples via SC-CO2 increased comparing

156 with the starting materials. Extraction pressure is a major factor affecting the β-carotene content

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157 and yield (Spanos, Chen, & SCHWARTZ, 1993). The results clearly indicated that the β-

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158 carotene content decreased with increasing pressure from 20 MPa to 30 MPa in Table 2

159 (comparing four sets data: No.1-3; No.4-6; No.7-9; No.10-12), when the extraction temperature

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160 and time were fixed. It is because the solubility of β-carotene increased and more β-carotene was

161 extracted too with increasing pressure. Therefore, the β-carotene content concentration did not

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increase in the extract with increasing pressure from 20MPa to 30MPa. So, the pressure of 20
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163 MPa is suitable regarding the β-carotene content. When the extraction pressure and the extraction

time were fixed at 20 MPa and 2 hours, respectively, the β-carotene content increased slightly,
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165 and the yield reduced a little with the increasing temperature, as shown in No.1 and No.7 in
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166 Table 2. The increase in temperature usually reduces SC-CO2 solvent power resulting in better
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167 selectivity of SC-CO2 to the β-carotene and impurity. Comparing the results of No.1 with No.4

168 and No.7 with No.10, it is found that the β-carotene content increases with the extending of
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169 extraction period, while the yield reduces. This can be attributed to the reason that more impurity

170 fractions were extracted with the extension of extracting time. Then, the β-carotene content in the
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171 remaining of the extractor increases. Decreasing the yield at the same time indicates that more an
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172 amount of components were extracted with the increase of time. In summary, extraction pressure,

173 extraction temperature and extraction period have significant influence on the β-carotene content

174 during SC-CO2 pretreatment. The β-carotene can be concentrated to 16.7 g/100g when the

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175 extraction pressure, extraction temperature, and extraction time were 20 MPa, 60 °C, and three

176 hours, respectively.

177 3.3 Enriching β-carotene from the pretreated sample using n-hexane solvent extraction

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178 The conditions and results of the enriching β-carotene from the pretreated sample are listed in

179 Table.3. The pretreated β-carotene with 16.7 g/100g is selected as starting materials for

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180 concentration further by using n-hexane solvent extraction. The amount of the starting materials

181 was fixed as 4 g for each trial. As shown in Table 3, temperature and the ratio (mL:g) between

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182 hexane and the starting materials have significant influence on the β-carotene content. When the

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183 temperature was fixed, the β-carotene content increased with the increasing of the ratio (mL:g),
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184 such as No.1-4; No.5-8; No.9-12; No.13-16. This can be attributed to that more impurity

185 fractions were transferred into the hexane when its amount increased resulting in increasing the
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186 β-carotene content in the solid portion. Comparing the results of No.1-4 in Table 3, the β-

187 carotene content reached the maximum value, 40.2 g/100g, at the maximum ratio (No.4) when
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188 the temperature was -30℃. Comparing the results of No.1, No.5, No.9, No.13 in Table 3, we can
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189 find that the β-carotene content increased with increasing temperature when the ratio was fixed.

190 The β-carotene content reached 58.7 g/100g when the temperature and the rate were -5 °C and
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191 200 to 4, respectively, as shown in No.16 in Table 3. The digital photo of the β-carotene sample
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192 with 58.7 g/100g is shown in Fig. 3 from which it can be seen that the appearance and state of
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193 the β-carotene sample have completely been different compared with the starting materials. It is

194 a crystalline solid, while the starting materials (the pretreated β-carotene sample with 16.5

195 g/100g) is a kind of sticky paste

196 Though β-carotene solubility could reduce in the solvent with the decreasing temperature, the β-

197 carotene content was not the highest when the temperature was the lowest as shown in Table 3.

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198 One reason is that the too low temperature increased the appearance viscosity of the solution,

199 which prevented the β-carotene separation from other fractions. The other one is that more

200 amount of the impurity component would be deposited as their solubility in the solvent would

201 reduce with the reducing temperature, which would decrease the β-carotene content. The

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202 temperature induced two different effects. Therefore, the β-carotene content was not the highest

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203 at the lowest temperature. Selecting suitable temperature is critical to get high-content β-carotene

204 by using n-hexane solvent extraction.

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205 4. Conclusion

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206 A two-step approach, supercritical CO2 pretreatment followed by solvent extraction strategy, was
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207 demonstrated to enable to boost β-carotene concentrate with 10 g/100g made from crude palm oil

208 up to the desired high-content. The β-carotene was concentrated to 16.7 g/100g when the
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209 pretreatment was performed at conditions of 20 MPa, 60 °C, three hours and CO2 flow rate of 22
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210 kg/h, during which some impurity components interfering with separation process of solvent

211 extraction were removed. The pretreated β-carotene concentrate was boosted up to 58.7 g/100g
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212 from 16.7 g/100g by using n-hexane extraction when extraction conditions were as follow: the
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213 ratio between solvent and the pretreated sample is 200:4(mL/g), the processing temperature is at

214 -5°C and the centrifuge is at 860 xg. Supercritical CO2 pretreatment is an essential step to obtain
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215 a high-content β-carotene concentrate.

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216 Acknowledgments

217 We acknowledge Instrumental Analysis Center of SJTU for analysis.

218 Conflict of Interest: The authors declare that they have no conflict of interest

219

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220 References

221 Baysal, T., Ersus, S., & Starmans, D. (2000). Supercritical CO2 extraction of β-carotene and lycopene

222 from tomato paste waste. Journal of Agricultural and Food Chemistry, 48(11), 5507-5511.

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223 Choo, Y. M., Yap, S. C., Ooi, C. K., Ong, A. S. H., & Goh, S. H. (1992). Production of Palm Oil Carotenoid

224 Concentrate and its Potential Application in Nutrition. 243-254.

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225 Eckey, E. W. (1949). Process for preparing carotenoid concentrates from palm oil. In): Google Patents.

226 Heidlas, J., Huber, G., Cully, J., & Kohlrausch, U. (1998). Process for the extraction of carotenes from

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227 natural sources. In): Google Patents.

228 Holick, C. N., Michaud, D. S., Stolzenberg-Solomon, R., Mayne, S. T., Pietinen, P., Taylor, P. R., Virtamo,

229
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J., & Albanes, D. (2002). Dietary carotenoids, serum beta-carotene, and retinol and risk of lung
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230 cancer in the alpha-tocopherol, beta-carotene cohort study. Am J Epidemiol, 156(6), 536-547.
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231 Johnson, E. J. (2002). The Role of Carotenoids in Human Health. Nutrition in Clinical Care, 5(2), 56-65.

232 Khoo, L., Morsingh, F., & Liew, K. (1979). The adsorption of β-carotene I. by bleaching earths. Journal
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233 of the American Oil Chemists Society, 56(7), 672-675.

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234 Latip, R., Baharin, B., Man, Y. C., & Rahman, R. A. (2001). Effect of adsorption and solvent extraction

235 process on the percentage of carotene extracted from crude palm oil. Journal of the American
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236 Oil Chemists' Society, 78(1), 83-87.

237 Ooi, C., Choo, Y., Yap, S., Basiron, Y., & Ong, A. (1994). Recovery of carotenoids from palm oil. Journal
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238 of the American Oil Chemists’ Society, 71(4), 423-426.

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239 Puah, C., Choo, Y., Ma, A., & Chuah, C. (2005). Supercritical fluid extraction of palm carotenoids.

240 American Journal of Environmental Science, 1(4), 264-269.

241 Spanos, G. A., Chen, H., & SCHWARTZ, S. J. (1993). Supercritical CO2 Extraction of β‐Carotene
β‐ from

242 Sweet Potatoes. Journal of Food Science, 58(4), 817-820.

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243 Tan, B., & Saleh, M. H. (1992). Integrated process for recovery of carotenoids and tocotrienols from

244 oil. In): Google Patents.

245 Zaidul, I., Norulaini, N. N., Omar, A. M., & Smith, R. (2007). Supercritical carbon dioxide (SC-CO 2)

246 extraction of palm kernel oil from palm kernel. Journal of Food Engineering, 79(3), 1007-1014.

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263 Figures captions

264 Fig 1. Schematical drawing of enriching beta-carotene

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265 Fig 2. 1. CO2 cylinder, 2.Filter, 3. Condenser, 4.CO2 pump, 5. Pressure back regulator, 8, 11, 14

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266 Pressure gauge, 6, 9, 12.Heat exchanger, 7 Extractor, 10, 13. Separators

267 Fig 3. (a) Supercritical CO2 pretreated sample and (b) n-hexane solvent extracted β-carotene

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268 concentrated sample

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Table 1 Experimental conditions and results of enriching β-carotene from 10 g/100g β-carotene
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Run Solvent used (mL) Temperature (°C) % yield of solid fraction β-carotene content %

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1 50 -10 43.0± 0.63 11.4± 0.44

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2 100 -10 36.7± .71 11.9± 0.67

3 200 -10 32.2± 1.00 12.0± 0.56

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4 50 -20 41.0± 0.91 10.8± 0.58

5 100 -20 33.7± 1.06 12.5± 0.62

6 200 -20
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7 50 -30 38.7 ± 0.81 11.7± 0.56

8 100 -30 35.0± 0.52 12.3± 0.68

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9 200 -30 30.5± 0.84 12.0± 0.36

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Table 2 SC-CO2 pretreated conditions and results

Run Pressure Temperature Time % yield β-carotene

(MPa) (°C) (h) (%)

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1 20 60 2 65.2 ± 1.10 15.3± 0.90

2 25 60 2 45.0± 0.85 14.5 ± 1.11

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3 30 60 2 33.7± 0.98 13.0± 1.01

4 20 60 3 55.6± 1.05 16.7± 0.74

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5 25 60 3 40.0± 0.90 14.0± 0.94

6 30 60 3 26.0± 0.51 13.0± 1.03

7 20 70
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8 25 70 2 40.0± 0.89 14.0± 0.76
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9 30 70 2 26.2 ± 0.76 12.0± 0.36

10 20 70 3 55.0 ± 0.91 16.4± 0.88

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11 25 70 3 39.3± 0.81 14.0± 1.03

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12 30 70 3 25.0± 1.05 12.0± 0.97

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Table 3 Conditions and results of enriching β-carotene from the pretreated sample
Run Solvent used Temperature % yield of solid β carotene content %
(mL) (°C) fraction

1 50 -30 24.3 ± 0.96 31.0 ± 1.07

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2 100 -30 23.0 ± 0.76 35.0 ± 1.01

3 150 -30 22.5 ± 1.01 37.3 ± 1.04

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4 200 -30 22.3 ± 0.73 40.2 ± 0.88

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5 50 -15 31.0 ± 1.17 33.7 ± 0.75

6 100 -15 24.0 ± 0.81 31.8 ± 1.16

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7 150 -15 23.3 ± 0.95 38.0 ± 0.97
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8 200 -15 22.0 ± 0.98 42.7 ± 1.02

9 50 -10 26.5 ± 1.16 37.5 ± 1.00

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10 100 -10 20.3 ± 0.88 51.0 ± 0.74

11 150 -10 19.0 ± 0.75 53.8 ± 0.94

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12 200 -10 19.0 ± 0.83 54.0 ± 1.18

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13 50 -5 22.0 ± 0.72 39.4 ±0.81

14 100 -5 19.5 ± 0.71 54.2 ± 0.81

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15 150 -5 18.5 ± 1.05 55.3 ± 1.01

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16 200 -5 18.0 ± 0.88 58.7 ± 0.89

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17 50 0 21.8 ± 1.05 39.0 ± 1.01

18 100 0 20.5 ± 0.77 44.3 ± 1.03

19 150 0 19.0 ± 0.97 49.7 ± 1.06

20 200 0 18.5 ± 1.00 52.0 ± 0.72

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Highlights

• A two-step method was developed to enrich beta-carotene from 10 g/100g beta-

carotene concentrate.

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• Supercritical CO2 extraction was an effective way to remove impurities like oil and

wax fraction.

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• Solvent extraction further boosted the pretreated beta-carotene concentrate from 16.5

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g/100g to 58.7 g/100g

• Extraction and purification mechanisms by supercritical CO2 and solvent extraction

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were explained.
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