3.2. Supercritical carbon dioxide extraction of bioactive compounds: a case study

The experimental strategy used for the supercritical carbon dioxide extraction process of bioactive compounds is based on the previous results collected by our research group in obtaining açai extracts [25].

Açaí is a dark purple, berry-like fruit from typical Amazon palm tree Euterpe oleracea Mart., integrated in the daily dietary habit of the native people.

Recently, many studies have suggested its use as a functional food or food ingredient due to its antioxidant activity, explained by the high content of phenolic compounds, such as anthocyanins, specially cyanidin-3-glucoside and cyanidin-3-rutinoside, flavones, and phenolic acids [26–28]. Phenolic constituents are generally associated with health-promoting properties and prevention of diseases [29–33]. Anthocyanins constitute a group of pigments, also important in the food industry, for the replacement of artificial colors [34–36].

The supercritical extraction experiments of the lyophilized açaí pulp under development were carried out in a Spe-ed™ SFE commercial unit (Allentown, PA, USA: model 7071 from Applied Separations) which is coupled to the solvent + co-solvent delivery system of Laboratory of Supercritical Extraction (LABEX), Faculty of Food Engineering-UFPA. The schematic representation of the supercritical extraction system is shown in Figure 5.

The first step consisted of the extraction with supercritical CO2 (pure) to obtain extracts rich in fatty acids and byproducts of the residual solid matrix (defatted pulp). Analyses of the content of bioactive compounds (anthocyanins and total phenolic compounds) were performed. The second stage that is under development consists of the extraction with supercritical CO2 combined with water as co-solvent applied to the residual solid matrix to obtain extracts concentrated in anthocyanins.

are separated by expansion (depressurizing), since at low pressures the density of the solvent sharply decreases, therefore it decreases the solubilizing power of the solvent as well and the

Figure 4. Scheme of a supercritical fluid extraction plant applying solvent/co-solvent. CO2 cylinder (1); cooling bath (2); booster (CO2 pump-3 and Compressor-4); mixer (5); CO-solvent pump (6); co-solvent recipient (7); extraction unit (8);

The choice of the operating condition (P and T) is a determining factor that contributes to the maximization of the extracts solubility in the supercritical solvent, and consequently the extraction yields. Thus, increasing the density of the supercritical fluid, the solubility of the solvent maximizes. The solubility increasing can also occur when a co-solvent is added, which

To design a high-pressure fluid extraction process of valuable compounds from new natural solid matrices, it is necessary to define the size of the extraction unit and some important parameters have to be determined to obtain the optimum process conditions for each application. Brunner

changes the solvent power and, in this way, the new solvent is a mixture [19, 20].

products precipitate.

control valve (V-5); separation vessel (9); flow meter (10).

216 Carbon Dioxide Chemistry, Capture and Oil Recovery

In the first stage, Batista et al. [25] subjected samples of lyophilized açaí pulp to the supercritical carbon dioxide extraction process. Among the results, the study of the process variables (temperature, pressure, and solvent density) that maximize the extraction yield of açaí oil, the quantification of the total anthocyanins content and total phenolic compounds content, and the evaluation of the allelopathic potential of the extracts obtained can be highlighted.

Figure 6 shows the experimental results of the 50, 60, and 70C isotherms on dry basis and their standard deviations. In this study, the highest global yield was equal to 45.4 0.58%,

obtained at 70C and 490 bar, while the lowest global yield was equal to 9.07 0.6%, obtained at 60C and 190 bar. The density is related to the CO2 solubility and is directly influenced by temperature and pressure. Here, the most important parameter was the density, since when it

The analysis of the phenolic compounds in the lyophilized açaí berry pulp showed an increase in its content comparing the samples before and after the extraction with supercritical CO2, in different conditions. Its highest content was equal to 7565 mg/100 g and was obtained in the condition of 70C and 350 bar. The standard deviation for each condition was lower than 0.18% (Figure 7). Regarding anthocyanins, there was also an increase in its content. Before the extraction with supercritical CO2, the total concentration was equal to 96.58 0.11 mg/100 g, and after the extraction, it reached up to 137.5 mg/100 g of sample in the condition of 50C and 220 bar. The standard deviation was lower than 0.15%. Figure 8 shows the values obtained and their specific deviation. It can be inferred that since the extracts of the lyophilized acaí berry pulp obtained by supercritical CO2 are rich in phenolic compounds and anthocyanins, it pre-

The results of the fatty acid profile analysis of açaí extracts indicate a low saturated/unsaturated ratio except for the condition of 70C and 320 bar. The SFA content reached 99.67% at the

220 bar

LABP 700 800 900

Figure 7. Total phenolic compounds content in lyophilized açaí berry pulp before and after extraction with supercritical

 **density (Kg/m3**

**)**

**CO2**

270 bar

320 bar

350 bar

420 bar

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219

490 bar

increased (in all isotherms), the oil global yield also increased.

sents great potential in nutraceutical applications.

150 bar

190 bar

220 bar

condition of 70C and 320 bar.

0

CO2. ( ) 50C, ( ) 60C, and ( ) 70C isotherms [17].

1000

2000

3000

4000

5000

**Phenolic Compunds Content (mg/100g)**

6000

7000

8000

9000

Figure 5. Experimental protocol for the bioactive compounds extraction.

Figure 6. Global yield on dry basis versus density of supercritical CO2 extraction of lyophilized açaí berry oil. ( ) 50C, ( ) 60C, and ( ) 70C isotherms [17].

obtained at 70C and 490 bar, while the lowest global yield was equal to 9.07 0.6%, obtained at 60C and 190 bar. The density is related to the CO2 solubility and is directly influenced by temperature and pressure. Here, the most important parameter was the density, since when it increased (in all isotherms), the oil global yield also increased.

The analysis of the phenolic compounds in the lyophilized açaí berry pulp showed an increase in its content comparing the samples before and after the extraction with supercritical CO2, in different conditions. Its highest content was equal to 7565 mg/100 g and was obtained in the condition of 70C and 350 bar. The standard deviation for each condition was lower than 0.18% (Figure 7). Regarding anthocyanins, there was also an increase in its content. Before the extraction with supercritical CO2, the total concentration was equal to 96.58 0.11 mg/100 g, and after the extraction, it reached up to 137.5 mg/100 g of sample in the condition of 50C and 220 bar. The standard deviation was lower than 0.15%. Figure 8 shows the values obtained and their specific deviation. It can be inferred that since the extracts of the lyophilized acaí berry pulp obtained by supercritical CO2 are rich in phenolic compounds and anthocyanins, it presents great potential in nutraceutical applications.

The results of the fatty acid profile analysis of açaí extracts indicate a low saturated/unsaturated ratio except for the condition of 70C and 320 bar. The SFA content reached 99.67% at the condition of 70C and 320 bar.

Figure 5. Experimental protocol for the bioactive compounds extraction.

150 bar

0

60C, and ( ) 70C isotherms [17].

10

20

30

**Global Yield on Dry Basis (%)**

40

50

60

218 Carbon Dioxide Chemistry, Capture and Oil Recovery

190 bar

220 bar

220 bar

270 bar

700 800 900

 **density (Kg/m3**

**)**

**CO2**

Figure 6. Global yield on dry basis versus density of supercritical CO2 extraction of lyophilized açaí berry oil. ( ) 50C, ( )

320 bar

350 bar

420 bar

490 bar

Figure 7. Total phenolic compounds content in lyophilized açaí berry pulp before and after extraction with supercritical CO2. ( ) 50C, ( ) 60C, and ( ) 70C isotherms [17].

Figure 8. Total anthocyanins compounds content in lyophilized açaí berry pulp before and after extraction with supercritical CO2. ( ) 50C, ( ) 60C, and ( ) 70C isotherms [17].

where

Table 1. Bender equation constants for CO2 [19].

national Thermodynamic Table.

ð1Þ

221

ð2Þ

ð3Þ

ð4Þ

ð5Þ

ð6Þ

ð7Þ

ð8Þ

ð9Þ

Figure 9 shows the calculation with Bender equation [19] of state for the P-V-T diagram isotherms and saturation curve, including an isotherm close to the critical temperature of the carbon dioxide. The calculations were performed using a Microsoft Excel spreadsheet. The equation presents accuracy in calculations when compared to data taken from IUPAC Inter-

i ai i ai

 0.22488558 11 0.12115286 0.13717965 � 103 <sup>12</sup> 0.10783386 � <sup>10</sup>�<sup>3</sup> 0.14430214 � 105 <sup>13</sup> 0.43962336 � <sup>10</sup>�<sup>2</sup> 0.29630491 � 107 <sup>14</sup> �0.36505545 � <sup>10</sup><sup>8</sup> 0.20606039 � 109 <sup>15</sup> 0.19490511 � 1011 0.45554393 � <sup>10</sup>�<sup>1</sup> <sup>16</sup> �0.29186718 � <sup>10</sup><sup>13</sup> 0.77042840 � <sup>10</sup>�<sup>2</sup> <sup>17</sup> 0.24358627 � 108 0.40602371 � 105 <sup>18</sup> �0.37546530 � <sup>10</sup><sup>11</sup> 0.40029509 <sup>19</sup> 0.11898141 � 1014 �0.39436077 � <sup>10</sup>�<sup>3</sup> <sup>20</sup> 0.50000000 � 101

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