Composition, thermal behavior and antioxidant activity of pracaxi (Pentaclethra macroloba) seed oil obtained by supercritical CO2

Journal Pre-proof Composition, thermal behavior and antioxidant activity of pracaxi (Pentaclethra macroloba) seed oil obtained by supercritical CO2 Gerson Lopes Teixeira, Laércio Galvão Maciel, Simone Mazzutti, Cintia Bernardo Gonçalves, Sandra Regina Salvador Ferreira, Jane Mara Block PII:

S1878-8181(20)30039-6

DOI:

https://doi.org/10.1016/j.bcab.2020.101521

Reference:

BCAB 101521

To appear in:

Biocatalysis and Agricultural Biotechnology

Received Date: 6 January 2020 Revised Date:

31 January 2020

Accepted Date: 31 January 2020

Please cite this article as: Teixeira, G.L., Maciel, Laé.Galvã., Mazzutti, S., Gonçalves, C.B., Salvador Ferreira, S.R., Block, J.M., Composition, thermal behavior and antioxidant activity of pracaxi (Pentaclethra macroloba) seed oil obtained by supercritical CO2, Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2020.101521. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Contributions Section CRediT author statement for the manuscript entitled: “Composition, thermal behavior and antioxidant activity of pracaxi (Pentaclethra macroloba) seed oil obtained by supercritical CO2”

Authors:

Gerson Lopes Teixeira: Conceptualization, Methodology, Formal analysis, Investigation, Writing - Original Draft, Visualization. Laércio Galvão Maciel: Investigation. Simone Mazzutti: Investigation, Writing - Review & Editing. Cintia Bernardo Gonçalves: Investigation, Writing - Review & Editing. Sandra Regina Salvador Ferreira: Conceptualization, Methodology, Resources, Writing - Review & Editing. Jane Mara Block: Conceptualization, Methodology, Resources, Supervision, Project administration, Funding acquisition, Writing - Review & Editing.

Pentaclethra macroloba

oil

SFE SC-CO2

seeds

flour

cake

Extraction yield

Triacylglycerols

Fatty acids

Thermal behavior

Phenolic compounds

Solid fat content

Antioxidant activity

Oxidative stability

Composition, thermal behavior and antioxidant activity of pracaxi (Pentaclethra macroloba) seed oil obtained by supercritical CO2

Gerson Lopes Teixeira a, Laércio Galvão Maciel a, Simone Mazzutti b, Cintia Bernardo Gonçalves c, Sandra Regina Salvador Ferreira b, Jane Mara Block a,*

a

Department of Food Science and Technology, Federal University of Santa Catarina,

88034-001, Florianópolis, SC, Brazil. b

Chemical and Food Engineering Department, Federal University of Santa Catarina,

88040-900, Florianópolis, SC, Brazil. c

Department of Food Engineering, Universidade de São Paulo, 13635-900, Pirassununga,

São Paulo, Brazil.

[email protected]

(GLT);

[email protected]

(LGM);

[email protected] (SM); [email protected] (CBG); [email protected] (SRSF); [email protected] (JMB).

_________________________________________ *Corresponding author: [email protected]

Abstract The Amazonian tree Pentaclethra macroloba (pracaxi) has pod-shaped fruits with flattened seeds mostly composed of lipids, carbohydrates, and proteins, besides secondary compounds, which provide potential use by many industries. This study evaluated the supercritical carbon dioxide (SC-CO2) extraction of pracaxi seed oil (PSO) at different conditions of pressure (200-300 bar) and temperature (40-60 °C). The PSO was assessed for its fatty acids and triacylglycerols (TAG) compositions, total phenolic content and antioxidant activity, as well as the oxidative stability and thermal behavior. SC-CO2 extractions provided oil yields (Ye) from 12 to 42 g/100 g, which represented up to 79% of the Ye obtained with hexane (Soxhlet). The pressure was the only significant variable (p < 0.05) on SC-CO2 Ye. In general, PSO showed a fatty acid profile rich in oleic, behenic, and linoleic acids (53.46, 16.54%, 12.22%, respectively), which reflected in the TAG structure mostly composed of Oleic-Oleic-Oleic, Oleic-Oleic-Behenic, and Oleic-Oleic-Linoleic (20.24, 16.10, and 13.21%, respectively). PSO samples also presented phenolic compounds (30.28-54.05 mg GAE/kg), and high stability to oxidation (11.38 h). The in vitro antioxidant activity was 0.935-2.273 µM Fe2+/kg for FRAP; 138.34-300.01 mg AAE/kg for CUPRAC; 21.81-41.03% inhibition for DPPH, and 8.60-24.84% inhibition of lipid peroxidation. DSC analysis showed a typical behavior for PSO with two major peaks under crystallization and one peak after melting, which revealed that this oil might present fat crystals at refrigeration temperature, but is liquid at 20 °C. The SC-CO2 represents an ecofriendly and sustainable approach for valorizing pracaxi seeds and obtaining highvalued coproducts.

Keywords: Pracaxi oil; Behenic acid; Supercritical fluid; Thermal properties; Antioxidant activity; Triacylglycerols.

1

1. Introduction

2

The Amazon rainforest, which is considered the largest in the world, corresponds

3

to more than half of the remaining rainforests. It concentrates the largest reserve of natural

4

products worldwide with immeasurable plant diversity and the potential for sustainable

5

exploitation of new raw materials. In addition, it is part of the Amazon biome, the largest of

6

the six Brazilian biomes. The conservation of the Amazon rainforest has been recently

7

debated internationally due to its dimension and ecological importance.

8

The Pentaclethra macroloba (Willd.) Kuntze tree, from the Fabaceae family,

9

commonly known as “Gavilán tree,” “pracaxi,” or “pracachy” is an Amazonian tree that

10

has been gaining attention due to the high potential for eco-sustainable exploration. The

11

tree occurs naturally in many countries such as Brazil, Peru, Colombia, Venezuela,

12

Trinidad and Tobago, Guyana, and Suriname in South America, in addition to Nicaragua,

13

Honduras, Jamaica, Cuba, Costa Rica, and Panamá, in Central America (Fig. 1a-b) (EOL,

14

2019; Orwa et al., 2009). The P. macroloba produces a pod-shaped fruit (20-25 cm) that

15

contains between 3 to 8 seeds (see detail in Fig. 1c-f). These edible seeds provide 45-48%

16

oil (Orwa et al., 2009) which is rich in monounsaturated fatty acids such as oleic acid (47.3-

17

53.5%), also presenting expressive content of behenic acid (16.1-25.5%), followed by

18

linoleic (11.7-13.1%) and lignoceric (12.5%) acids (Bezerra et al., 2017; Costa et al., 2014;

19

Pereira et al., 2019; Teixeira et al., 2012). Mostly obtained from small industries in Brazil,

20

some companies sell raw pracaxi oil worldwide in online stores for about 50-75 USD per

21

liter. This oil is generally used as an ingredient by the national and international cosmetics

22

industry in oil blends (with coconut, olive, andiroba, argan, and açaí oils), soaps,

23

moisturizing, exfoliating, skin cleaner, conditioner, and shampoos.

1

24

The oil from pracaxi seeds has been proven to show healing effects for scar and

25

wound (Banov et al., 2014), and action against insects correlated to the content of phenolics

26

compounds and other secondary metabolites (Santos et al., 2016). In addition, the

27

physicochemical, chemical and thermal properties (Costa et al., 2014; Lima et al., 2017),

28

the ability to inhibit enzymatic activity (Teixeira et al., 2012), the cytotoxicity and

29

genotoxicity (Maistro et al., 2013) of cold-pressed pracaxi oil have also been reported.

30

Despite its main utilization for medicinal and cosmetic purposes, pracaxi oil is also used as

31

a frying oil by riverside populations in the Brazilian Amazon region (Crespi and Guerra,

32

2013). However, some of its properties remain unknown.

33

The traditional extraction techniques to obtain pracaxi oil requires cooking the

34

seeds prior to the extraction (Crespi and Guerra, 2013), while the industrial process is

35

performed by hydraulic presses. Nevertheless, both methods yield low amounts of oil, and

36

the resulting cake is discarded with significant contents of oil, a problem that could be

37

solved by using more efficient extraction techniques. Currently, the valorization of oilseeds

38

using sustainable extraction techniques, mostly to obtain high-value products and

39

compounds with nutritional properties and/or bioactivity are encouraged. Supercritical

40

Fluid Extraction (SFE) is broadly reported as an environment-friendly technology, used

41

especially for obtaining oil-rich products with promising results (Catchpole et al., 2018;

42

Reverchon and De Marco, 2006). Carbon dioxide (CO2) is the typical solvent in SFE,

43

mainly because of the low cost, safety to handle, and availability. Furthermore, CO2 can be

44

employed at mild temperatures and is appropriate for use in food processing (Brunner,

45

2005; Reverchon and De Marco, 2006; Temelli, 2009). Besides the lipid compounds such

46

as fatty acids, tocopherols, and phytosterols, SFE provides the extraction of phenolic

2

47

compounds with no use of organic solvents nor generation of toxic waste. The damage

48

and/or loss of target compounds that are usually caused by conventional extractions using

49

high temperatures (e.g., Soxhlet) are also avoided (Reverchon and De Marco, 2006).

50

The chemical properties of pracaxi seeds and their extraction using pressurized

51

fluids have not been reported in the literature. Thus, in this study, a green-based extraction

52

using supercritical CO2 for obtaining oil from pracaxi seeds was evaluated and compared

53

with the traditional Soxhlet extraction using hexane. The yield of the extraction, as well as

54

the fatty acid profile, triacylglycerol composition, thermal behavior, total phenolic

55

compounds, and antioxidant capacity, were also studied.

56 57

2. Material and Methods

58 59

2.1. Sample

60

Pracaxi (Pentaclethra macroloba) seeds, from the 2018 harvesting, were kindly

61

donated by the company Amazon Oil (Ananindeua, PA, Brazil), which processes many

62

Amazonian oilseeds obtained under sustainable practices. The seeds were milled using an

63

IKA mill A11 (Campinas, SP, Brazil), and the obtained ground powder was sieved using a

64

#14 Tyler mesh (average size of 1.19 mm). Then, the powder was vacuum-packed, frozen,

65

and stored at −18 °C until the extractions.

66 67

2.2. Chemicals

68

Gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, 2,2′-

69

azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS), 2,4,6-Tri(2-

3

70

pyridyl)-s-triazine (TPTZ), and 6‐hydroxy‐2,5,7‐tetramethylchroman‐2‐carboxylic acid

71

(Trolox), were purchased from Sigma-Aldrich/Merck (São Paulo, Brazil). The other

72

reagents of analytical grade were obtained from Neon Comercial (São Paulo, Brazil).

73 74

2.3 Characterization of pracaxi seeds

75

The physical properties of the pracaxi seeds were assessed as follows: the average

76

weight and 100-seed weight were obtained using an analytical scale, while height, width,

77

and thickness were measured using a pachymeter (n=100). The moisture (oven at 105 °C),

78

lipids (Soxhlet), total Nitrogen (Kjeldahl), and ash content were assessed according to

79

official methods (AOAC, 2007). Proteins were estimated using a conversion factor of 5.30.

80

The carbohydrates were calculated by difference. CIELab color parameters were measured

81

in a CR-400 colorimeter Konica Minolta Sensing Americas (Ramsey, NJ, USA).

82 83

2.4 Supercritical fluid extraction (SFE)

84

The apparatus used in the supercritical extractions was detailed by Mazzutti et al.

85

(2018). The setup comprised a CO2 reservoir, a thermostatic bath, an M111 high-pressure

86

booster (Maximator, Germany), and a stainless-steel jacketed extractor with 138.2 mL of

87

internal volume (internal diameter of 20 mm and 440 mm of height). The extraction cell

88

was also connected to a thermostatic bath in order to control the extraction temperature.

89

The pressure and flow rate were controlled with the aid of high-pressure valves, flowmeter,

90

and analogical manometers. High-purity carbon dioxide (99.9%, White Martins, Brazil)

91

was the supercritical solvent used.

4

92

The extraction column was fed with 15 g of the raw material, while the empty

93

space was filled with dried cotton and glass beads. Aiming to evaluate the impact of

94

temperature (40-60 °C) and pressure (200-300 bar) on the extraction yield (Ye), the

95

extractions followed a 2² factorial design with three replications at the center point,

96

totalizing 7 runs (Table 1). The extraction time and extraction kinetics were established at a

97

pre-set condition (40 °C and 250 bar), allowing the definition of the 240 min of extraction

98

for the yield assays, after reaching the diffusional step as shown in the kinetic curve

99

depicted in Fig. 2a. A constant flow rate of 0.7 kg of CO2 per hour was used. The oil

100

sample was collected in amber glass vessels. The Ye of the pracaxi seed oil (PSO) was

101

estimated as the % of the mass of oil (Moil) from the total mass of raw material fed (Mrm) as

102

the Eq. (1). % =

∗ 100

(1)

103

A control oil sample was extracted by Soxhlet during 360 min with 150 mL

104

hexane (99.9%) and 5 g of pracaxi seeds. After the extraction, the solvent was removed at

105

50 °C under vacuum in a rotary evaporator (Model 801, Fisatom Ltda., Brazil). All the oil

106

samples from SFE and Soxhlet were stored at −18 °C until the analysis.

107 108

2.5. Fatty acid profile and Triacylglycerol composition of pracaxi oil

109

The fatty acids of PSO were determined in a gas chromatograph Shimadzu GC-

110

2014 (Kyoto, Japan). The setup has a flame ionization detector (FID) and a split/splitless

111

injector. The carrier gas was Nitrogen (99.99%) flowing at 1.10 mL/min. The methyl esters

112

(FAME) were obtained following a methodology described by Hartman and Lago (1973).

5

113

A 105-meter capillary column Restek RTX- 2330 (Bellefonte, PA, USA) with 0.25 mm i.d.

114

and 0.20 µm df was used. The run started and kept at 140 °C for 5 min, and then increased

115

(2.5 °C/min) up to 240 °C and held for 15 min. The injector (260 °C) and detector (280 °C)

116

were kept at constant temperatures. Fatty acids were reported as % of the total peak area, as

117

compared with a Supelco FAME Mix standard.

118

The probable triacylglycerol (TAG) composition of the pracaxi seed oil was

119

assessed based on the fatty acid profile with the aid of a statistical algorithm created by

120

Antoniosi Filho et al. (1995) by a random distribution using the MATLAB R2015a (The

121

MathWorks, Inc., Natick, MA USA).

122 123

2.6 Differential Scanning Calorimetry (DSC) analysis

124

The thermal behavior of PSO was evaluated in a DSC Perkin Elmer 8500

125

(Waltham, MA, USA) calibrated with Indium, under 20 mL/min nitrogen flow with

126

99.99% purity (White Martins, Araucária, PR, Brazil). About 8 mg of oil sample was used,

127

and the crystallization and melting behaviors were investigated. The oil remained at 20 °C

128

for 5 min, and then it was heated to 50 °C (5 °C/min) and kept for 5 min. Afterward, the

129

sample was cooled at 5 °C/min to −80 °C and held for 5 min. Finally, the oil was heated

130

from −80 °C to 50 °C (5 °C/min). The peaks and enthalpies were calculated using Pyris™

131

software.

132

The solid fat content (SFC) was also estimated using Pyris™, and the curve was

133

plotted with the aid of Origin 8.6 (Originlab, Northampton, MA, USA) from the integrated

134

area under the melting curve.

135 6

136

2.7 Total phenolic compounds (TPC)

137

Previously to the analysis of TPC and antioxidant capacity, a procedure to obtain

138

methanolic extracts of PSO was performed with one gram of oil and 3 mL of 90% (v/v)

139

aqueous methanol solution as described by Bail et al. (2008).

140

The TPC were measured according to Singleton and Rossi (1965), with

141

modifications detailed by Granato et al. (2016). The absorbance was monitored at

142

λ = 725 nm. Ultrapure water was the blank. The results of triplicate evaluations were

143

reported as milligrams of gallic acid equivalent per kilogram of PSO.

144 145

2.8. In vitro reducing capacity and antioxidant activity

146

The same methanolic extracts used in Section 2.7 were used in the antioxidant

147

capacity assays. The scavenging activity of DPPH radical was assessed as recommended by

148

Brand-Williams et al. (1995). The absorbance at λ = 517 nm was registered, and ultrapure

149

water was used as a blank. The antioxidant activity, expressed as percentage scavenging of

150

DPPH (AA, %) was calculated as follows: AA = [1 – (Abssample /Absblank) × 100]. Ferric

151

Reducing Antioxidant Power (FRAP) analysis followed the method from Benzie and Strain

152

(1996). The absorbance of the PSO extract samples was monitored at λ = 593 nm, and the

153

results were expressed as µM Fe2+/kg PSO. The cupric reducing antioxidant capacity

154

(CUPRAC) assay was done as recommended by Apak et al. (2007) and expressed as

155

ascorbic acid equivalents per kg of PSO.

156

The in vitro antioxidant activity using chemical systems was compared to a

157

biological-based method that uses egg yolk and evaluates the inhibition of lipid

158

peroxidation (Margraf et al., 2016). The absorbance was read at λ = 532 nm. A control

159

sample was prepared using ultrapure water instead of the extracts. The result was calculated 7

160

as follows: % inhibition = (Abscontrol – Abssample/Abscontrol). All the antioxidant activity

161

assays were done with adaptations to microplates proposed by Margraf et al. (2016).

162 163

2.9. Oxidative stability index (OSI)

164

The evaluation of the oxidative stability index (OSI) followed the AOCS method

165

Cd 12b-92 (AOCS, 1997) in a Rancimat 893 (Metrohm, Switzerland). One sample obtained

166

by the SFE at 40 °C and 300 bar was evaluated for its OSI based on the yield. The analysis

167

was performed using 3 g of pracaxi oil. The apparatus was set to run at 110 °C and 120 °C

168

with 20 L/h of airflow. The OSI was expressed as “hours” from duplicate runs.

169 170

2.10. Statistical analysis

171

The results were presented as the mean ± standard deviations. Significant

172

differences were detected by Duncan’s or t-test (p < 0.05). The effect of the temperature

173

and pressure on the oil yield was verified by ANOVA using Statistica 10.0 (StatSoft Inc.,

174

Tulsa, OK, USA) using a mathematical model as follows: =

+

+

+

175

where Ri is the response; β0 is a constant, and β1, β2 and β12 are the regression terms; T is

176

the temperature, and P is the pressure.

177 178

3. Results and Discussion

179

3.1 Characterization of pracaxi seeds

180

Table 2 shows the physical and physicochemical properties of pracaxi seeds. The

181

average weight of a pracaxi seed was 6.36 g, while one hundred seeds account for

182

approximately 635 g. The width, length, and thickness were 31.19 mm, 40.25 mm, and 9.60 8

183

mm, respectively. The lipid content (53 g/100 g) was higher than that described by Orwa et

184

al. (2009). The carbohydrates represented 25 g/100g of the seeds, followed by 15 g/100 g of

185

proteins, moisture (4 g/100 g), and ash (<2 g/100 g). The color of pracaxi seeds varies from

186

light yellow to dark brown (Fig. 1). On the other hand, the ground seeds of pracaxi showed

187

a brownish color since the L parameter (lightness) tended to the center of the scale, while a

188

and b parameters showed a tendency to red and yellow, respectively. These results were

189

also supported by both chroma and hue angle parameters.

190 191

3.2 SFE Kinetics

192

An extraction kinetics curve was obtained by SFE with CO2 at 250 bar and 40 °C

193

to establish the extraction time for the SC-CO2 yield assays. Fig. 2a shows the SFE curve

194

for the pracaxi seeds representing the mass of oil extracted versus extraction time. In the

195

first 240 min of extraction, approximately 36 wt% yield was achieved, while between 240

196

and 360 min of extraction, the recovery of oil presented only a small increase (<1.5 wt%).

197

Thus, the time of the subsequent extractions was set in 240 min. In the SC-CO2 extraction,

198

the easily accessible oil content was firstly extracted because it surmounts only the

199

diffusion resistance in the carbon dioxide. Usually, the SFE using this solvent presents

200

three stages, i.e., the constant extraction rate, falling extraction rate, and the diffusion-

201

controlled extraction rate (Sovová, 1994). Fig. 2a shows that the extraction of pracaxi seed

202

oil presented these three steps.

203

The experimental parameters applied in the SFE of pracaxi seeds, the Ye, as well

204

as the density and viscosity of the CO2 are shown in Table 1. After exposing the raw

205

material to the compressed CO2 flow for 240 min under different conditions, the yield of 9

206

the SFE extraction ranged from 12 to 42 g oil/100 g. On the other hand, a yield of 53.42 ±

207

1.75 g/100 g was obtained after 360 min of extraction using hexane as a solvent in the

208

Soxhlet system. The longer extraction time, higher temperature, solvent renewal, as well as

209

the hexane polarity, may have positively influenced the higher Ye obtained by Soxhlet

210

when compared to the SFE. Similar Ye obtained by Soxhlet can be accomplished by SFE if

211

more extended periods of time for the complete exhausting of the sample, or if higher

212

working pressures are applied (Cunha et al., 2019; Nimet et al., 2011).

213

Although the yield was higher for the Soxhlet extraction, after vacuum

214

evaporation, traces of the solvent may remain in the oil. On the other hand, SFE provides a

215

completely solvent-free extract with no need for further evaporation. Also, the use of

216

supercritical CO2 as a solvent to lipid extraction is interesting because there have been

217

growing concerns regarding the use of hexane in extraction processes. Besides the fact that

218

it is petroleum-derived, hexane has been listed by the Environmental Protection Agency

219

(EPA) as a hazardous air pollutant in the Clean Air Act in 1990 (DeSimone, 2002). In

220

pharmaceutical and nutraceutical products, hexane and cyclohexane are classified as class 2

221

solvents, which refers to solvents that should have minimal use and presence in the material

222

due to possible causative agents of irreversible toxicity (Kerton and Marriott, 2013).

223

It was observed up to 79% of the efficiency of extraction (Ee) for SFE when

224

compared to the Soxhlet system. Corso et al. (2010) and Pederssetti et al. (2011) reported a

225

yield of extraction of lipids for sesame (35 wt% at 40 °C/250 bar) and canola (19 wt% at 40

226

°C/250 bar) when compared to Soxhlet, which reached 53% and 69% Ee, respectively.

227

Oliveira et al. (2019) reported a lower Ee (57 wt% at 60 °C/250 bar) using SFE for babassu

228

seed than the Soxhlet method, which provided an Ee of 86%. Other studies with SC-CO2

10

229

reported 100% Ee on the oil extraction from bacaba pulp (46 wt% at 60 °C/270 bar) and

230

sunflower seeds (41 wt% at 40 °C/250 bar) (Cunha et al., 2019; Nimet et al., 2011).

231

The SFE showed to be a reliable, robust, and fast method for extracting those oils.

232

The differences in the results reported in the literature are associated with the operational

233

conditions, extraction time, and the nature of the raw materials. In addition to the shorter

234

extraction time in SFE, no further steps to filter the oil are required, and no residual solvent

235

is remaining, once the CO2 is completely removed after the pressure release in the

236

extraction cell. The advantages of SC-CO2 also include the ability to preserve the flavor

237

and aroma, recover bioactive compounds such as tocopherols, phytosterols, and omega-3

238

fatty acids, which are well-preserved because the oil is obtained in an oxygen-free

239

environment (Van Hoed, 2010). The phospholipids are also preserved as they are insoluble

240

in CO2 (Catchpole et al., 2018; Temelli, 2009). Therefore, in some cases, as the extraction

241

of fruit and nut oils, there is no need for refining steps, and the oil obtained, which is rich in

242

bioactive compounds, is generally ready to be applied in foods, pharmaceuticals, or

243

cosmetics (Van Hoed, 2010).

244

In this study, after the final extraction time (240 min), the solvent to feed mass

245

ratio was ≈ 187 g CO2/g pracaxi seeds, which may be a drawback in larger scales. There are

246

many ways to solve this issue and reduce the consumption of CO2. For instance, using the

247

so-called ultra-high pressure CO2 extraction (up to 1300 bar), increasing the solvent flow,

248

reducing the particle size of the sample, using co-solvents (as ethanol), enzymes, or

249

combining other emerging techniques as the case of ultrasound probes in the SFE system

250

may provide several enhancements in the extraction processes, diminishing the

11

251

consumption of the solvent (Catchpole et al., 2018; Reverchon and De Marco, 2006;

252

Temelli, 2009).

253 254

3.3 Effect of SFE conditions on the extraction yield

255 256

Adjustment of the extraction conditions is often necessary to obtain maximum

257

extraction efficiency. Additionally, mathematical modeling helps to verify different

258

interactions from the variables on the response, besides scaling-up laboratory procedures to

259

industrial levels (Reverchon and De Marco, 2006). The response surface for the Ye of

260

pracaxi seeds (Fig. 2b) and the Pareto chart (Fig. 2c) highlight the effects of pressure and

261

temperature on the dependent variable. The results show that the pressure (200-300 bar)

262

had a significant (p < 0.05) effect on the Ye. The higher the pressure, the higher the

263

extraction yield of pracaxi oil. In contrast, the temperature evaluated (40-60 °C) had no

264

significant influence on the response variable. In addition, increasing pressure in 100 bar

265

resulted in a more than 56% increase in the Ye, as also reported by other authors (Corso et

266

al., 2010; Cunha et al., 2019; Pederssetti et al., 2011). However, at low pressures as 200

267

bar, increasing temperature from 40 to 60 °C may decrease 8% of oil recovery (Table 1).

268

These differences are associated with the variations of viscosity and density of CO2, which

269

directly affect the lipid solubility in the extraction system. In fact, the density of the fluid is

270

a crucial factor that plays an essential role in the extraction of oil in the SFE (Temelli,

271

2009). Moreover, the chemical structure of the solute has to be considered, as well as the

272

chain length, molecular weight, the degree of unsaturation, and the occurrence of functional

12

273

groups (Yu et al., 1994), the particle size and residence time, along with the vapor pressure

274

of the sample (Reverchon and De Marco, 2006).

275

The density of CO2 is relatively lower at 60 °C and 200 bar (P2) in relation to the

276

other extraction conditions (Table 1), which may explain the lowest oil recovery under this

277

extraction condition. Similar behavior was described for the SC-CO2 extraction of canola

278

seed oil at the same conditions (Pederssetti et al., 2011). In addition, low oil recovery was

279

observed for sunflower (Nimet et al., 2011) and sesame (Corso et al., 2010) seeds oils, both

280

at 60 °C and 190 bar. This may be due to the low apparent solubility of the triacylglycerols

281

(TAGs) from those raw materials in SC-CO2 at pressures lower than 200 bar, since this

282

property is strongly correlated to the density of CO2, that in turn is affected by the system

283

pressure. On the other hand, after increasing the pressure (>200 bar), those authors reported

284

improvement in the oil extraction, confirming a stronger influence of pressure on the Ye.

285

Likewise, the highest Ye of PSO was observed for the sample P3, which was submitted to

286

the conditions of supercritical CO2 at the highest density (909.89 kg/cm3) and viscosity

287

(93.83 µPa s) (Table 1).

288

The interaction between temperature and pressure was not significant (p < 0.05),

289

while the temperature alone had a negative but not significant impact on the Ye of PSO

290

(Fig. 2c). On the other hand, the pressure showed a positive and significant (p < 0.05)

291

effect on the supercritical carbon dioxide extraction of PSO. This was confirmed by the

292

ANOVA (Table S1), which also demonstrates that the linear model (Ri = 29.1713 −

293

1.2264T + 0.0545P + 0.0039TP) was significant and demonstrated the influence of pressure

294

on the selectivity of extraction of the lipid fractions from pracaxi seed. Although not

295

significant in the Pareto chart, the effect of temperature on Ye is more adverse. At low 13

296

pressures the CO2 solubility decreases with increasing temperature as the specific solvent

297

mass decreases rapidly with increasing temperature; at high pressures, the temperature-

298

specific mass changes are much more discrete, so the increase in vapor pressure caused by

299

the temperature increase becomes more important than the slight specific mass decrease

300

(Brunner, 2005).

301 302

3.4 Fatty acid profile and TAG composition of pracaxi seed oil

303

Table 3 shows that pracaxi oil is rich in unsaturated (66%) and saturated fatty

304

acids (33%). The main unsaturated fatty acid in PSO is the oleic acid (53%), while behenic

305

acid (16%) was found as the major saturated fatty acid. Other fatty acids, such as linoleic

306

(12%), lignoceric (11%), and stearic (2.6%), were found. Small differences in the fatty acid

307

profile were observed in the pracaxi oil extracted by SC-CO2 when compared to the sample

308

obtained with hexane. A similar fatty acid composition for pracaxi was described by Costa

309

et al. (2014) and Pereira et al. (2019) for samples obtained by a cold press. On the other

310

hand, Bezerra et al. (2017) showed lower content of oleic acid (47%) and higher contents of

311

behenic (22%) and arachidic (12%) acids, which may be related to the origin of the seed

312

samples. Teixeira et al. (2012) reported the lowest content of behenic acid and the highest

313

content of linoleic acid (5.0 and 25.5%, respectively) for pracaxi oil.

314

Fatty acids such as lauric, myristic, arachidic and erucic acids have been

315

previously found in pracaxi oil (Bezerra et al., 2017; Costa et al., 2014; Pereira et al., 2019;

316

Teixeira et al., 2012), which were not detected in our samples. Regarding the erucic acid, a

317

content between 0.82 to 1.6% was reported in such oils (Pereira et al., 2019; Teixeira et al.,

318

2012). The absence of this fatty acid is a positive result since its intake has been linked to

14

319

toxic effects in animal experiments. In addition, its presence in human plasma

320

phospholipids has been associated with a higher incidence of congestive heart failure, as

321

reported by the European Food Safety Authority (Knutsen et al., 2016). The high

322

oleic:linoleic ratio (> 100:1) of pracaxi oil is a decisive factor that may contribute to a high

323

shelf life (Pereira et al., 2019). This oil has been used in cosmetics such as shampoos,

324

conditioners, and moisturizers since it is the highest natural source of behenic acid ever

325

discovered. This fatty acid shows lubricant, emollient, and soothing properties, that are

326

assumed to help to restore the skin’s natural oils, improving the overall levels of hydration

327

(Banov et al., 2014). Because of this composition, pracaxi oil can be considered a premium

328

oil, which also contributes to the interest of the industry in developing products with

329

increased economic value.

330

The TAG composition of pracaxi oil was statistically predicted based on the fatty

331

acid composition, considering only the fatty acids that were higher than 0.5% (Table 4).

332

The content of trisaturated fatty acids calculated for both oils extracted by SC-CO2 and

333

Soxhlet with hexane was 4.0 wt%. As influenced by its fatty acid composition, rich in oleic,

334

behenic, and linoleic acids, the main TAGs determined in pracaxi oils were OOO (24.07-

335

20.24 wt%) OOBe (15.98-16.10 wt%), and OOLi (13.12-13.21 wt%), representing ≈49

336

wt% of the TAGs. From 21 TAGs identified, behenic acid was present in 10 TAG species,

337

showing that the pracaxi oil has a unique profile. The behenic acid may be used as a

338

chemical marker, making easy the identification of adulteration. According to Karupaiah

339

and Sundram (2007), native oils have typical stereospecificity of fatty acids in TAGs. The

340

C18:1 and C18:2 fatty acids (the main fatty acid in vegetable oils) are preferably in the sn-2

341

position in this kind of lipid matrix, which was confirmed in this research. As also shown in

342

Table 4, the TAG profile of the oil extracted by SC-CO2 under 40 °C and 300 bar was very 15

343

similar to that obtained by Soxhlet using hexane, suggesting that the method of extraction

344

has little impact in the TAG composition of pracaxi seed oil.

345 346

3.6 Total phenolic compounds (TPC), antioxidant activity (AA), and inhibition of lipid

347

peroxidation (ILP) of pracaxi oils

348

The results regarding the antioxidant activity and the TPC of pracaxi oil are

349

depicted in Fig. 3. The methanolic extracts of PSO presented total phenolics ranging from

350

31.92 to 54.05 mg GAE/kg oil, and significant differences (p < 0.05) between the samples

351

were observed. It was reported that phenolic acids are the major bioactive compounds

352

present in oils from seeds, along with flavonoids that contribute to slow oxidative processes

353

in the extracted oils (Van Hoed, 2010). Among the oils obtained by SC-CO2, samples P1

354

and P2, which were extracted at 200 bar and 40 and 60 °C, showed the highest content of

355

TPC. The phenolics from those samples were also statistically equal to those obtained by

356

Soxhlet using hexane (PS). The AA measured by FRAP, CUPRAC, and DPPH assays

357

observed in the samples P1 and P2 were the highest among the six samples. The results

358

indicate that the extraction using lower pressure may result in higher levels of bioactive

359

compounds and higher AA of pracaxi oil. The results of the CUPRAC assay suggest that

360

P1 and P2 may have the highest content of hydrophilic (phenolic compounds) and

361

lipophilic antioxidants (β-carotene and α-tocopherol) since the method is able to measure

362

both antioxidants (Apak et al., 2008). The percentage of DPPH radical scavenged by

363

antioxidants from PSO (up to 41%) was higher than that reported for corn (11.1%),

364

grapeseed (13.4%), soybean (17.4%), flax (19.3%), and similar to that of rice bran (23.7%),

365

and sunflower (23.9%) oils (Siger et al., 2008).

16

366

The ILP analysis, differently from DPPH and FRAP assays, simulates the

367

physiological conditions of in vivo lipid oxidation (Margraf et al., 2016). The percentage of

368

ILP varied from 8.60-24.84% of inhibition and showed that samples extracted at higher

369

pressure conditions (250 or 300 bar) presented the highest AA (Fig. 3). Inversely to the in

370

vitro assays, this result suggests that samples P3, P4, and P5 may have other compounds,

371

which show significant in vivo antioxidant properties, such as tocopherols and other minor

372

substances not investigated herein. Gustinelli et al. (2018) reported for bilberry oil,

373

recovered by SFE at 200 bar and 60 °C, a higher antioxidant activity than that obtained at

374

200 or 350 bar at 40 or 50 °C. The authors discussed that their results are associated with

375

the higher content of vitamin E obtained at 60 °C.

376 377

3.5 Oxidative stability of pracaxi oils

378

The PSO showed OSI values of 11.38 ± 0.50 h at 110 °C, and 10.83 ± 0.13 at 120

379

°C, which may indicate a long shelf life for the evaluated oil. This result is associated with

380

the high concentration of monounsaturated and saturated fatty acids and with the presence

381

of antioxidant compounds such as tocopherols and β-carotene. Velasco and Dobarganes

382

(2002) reported that minor compounds have a higher impact on OSI than the major

383

compounds as triacylglycerols. It was also reported that non-polar phenolic compounds

384

play a role against lipid peroxidation in SC-CO2 extracted oils, as the antioxidant capacity

385

is usually correlated with the TPC, thus contributing to the oxidative stability of the oil

386

(Van Hoed, 2010). Previous reports showed different OSI of 8.52 to 10.42 h (at 110 °C)

387

(Costa et al., 2014) and 5.55 h (at 130 °C) (Bezerra et al., 2017) for pracaxi oil. Different

388

results may be associated with the different chemical composition (antioxidant and/or pro-

389

oxidant compounds) and with the initial quality of the seeds (Christodouleas et al., 2015; 17

390

Siger et al., 2008). Similar values for OSI have been reported for other vegetable oils

391

obtained with SC-CO2 at 60 °C. Sapucaia nut (Lecythis pisonis) and egusi seed (Citrullus

392

lanatus sub Mucosospermus) showed an OSI of 10.17 h, and 9.29 h, respectively (Olubi et

393

al., 2019; Teixeira et al., 2018).

394 395

3.6 Thermal behavior and solid fat content of pracaxi oils

396

The thermograms showing both profiles of melting and crystallization during the

397

temperature ramp are shown in Fig. 4. The six pracaxi oil samples presented a similar

398

pattern during cooling and heating. The samples showed two distinct exothermic peaks on

399

the crystallization curve (Fig. 4a), and one endothermic peak and a shoulder on the melting

400

curve (Fig. 4b).

401

During cooling, the onset temperature, which indicates the beginning of the phase

402

transition, varied from 16.20 to −13.23 °C. The first peak showed the change from liquid to

403

solid and was observed at temperatures of 4.84 (P1) to 1.29 °C (P2). On the other hand, the

404

second peak occurred in the temperature range of −40.42 (P2) to −50.44 (P3). These two

405

peaks are related to the crystallization of TAGs, which starts to crystallize at the onset

406

temperature. The peak 1 may be related to the fraction that is mostly composed of saturated

407

fatty acid, as behenic and lignoceric acids, since saturated triacylglycerols crystallize at

408

higher temperatures as compared to the unsaturated ones. The second peak may be

409

attributed to the unsaturated oil fraction, like oleic and linoleic acids (Barba et al., 2013).

410

These results are in agreement with a previous report on the thermal behavior of pracaxi

411

seed oil (Pereira et al., 2019).

412

The onset temperature of the six pracaxi oil samples from the heating curve ranged

413

from 7.33-11.44 °C. However, the melting event started at approximately −30 °C for all the 18

414

samples, which also presented a shoulder in the region of −20 to −15 °C, that can be

415

attributed to the melting of the highly unsaturated fraction, mainly the triunsaturated TAG

416

(Augusto et al., 2012). The phase transition for the melting of the samples happened

417

between 11.90-14.68 °C, which is related to the saturated fatty acids, mainly di and

418

trisaturated fatty acids (Pereira et al., 2019). This event comprised a broad range of

419

temperature of about 50 °C, and enthalpies varying between 62.99 to 73.58 J/g. Most of the

420

PSO samples showed a similar behavior during melting, but samples P2 (200 bar, 60 °C)

421

and P5 (250 bar, 50 °C) exhibited a difference of about 2 °C in the onset, peak, and endset

422

temperatures in relation to the other samples.

423

The Solid Fat Content (SFC) is associated with the crystalline fat in a sample and

424

affects many physical and chemical properties such as spreadability, resistance to

425

oxidation, thickness, and flavor (Santos et al., 2014). As shown in Fig. 4b, pracaxi oils did

426

not present any thermal event from −80 to −40 °C. For this reason, SFC was estimated from

427

the DSC melting curves from −40 °C to 30 °C, which is the region of the melting peak

428

(Fig. 5). Slight changes were observed on the melting behavior of the different oil samples.

429

The samples P2 and P5 presented minor variations on the SFC, while the other ones

430

followed the same melting pattern. The SFC of the six samples presented a less than one

431

percent decrease in the range of −40 to −25 °C. A quickly drop started from −20 °C to up to

432

10 °C, when the remaining solid fat content was about 10%. A decrease in the SFC can be

433

observed from 12.50 °C and reached 0% SFC at 25 °C. These results indicate that pracaxi

434

seed oil may have some fat crystals at refrigeration temperature (between 4 and 10 °C), but

435

is entirely liquid in temperatures higher than 20 °C. This melting behavior is crucial for

436

good spreadability (Santos et al., 2014), and may facilitate pumping processes at industrial

437

facilities. The fatty acid composition, as well as the TAG profile, have a straight impact on 19

438

the SFC because the oils containing higher unsaturated fatty acids content tend to melt

439

faster than the oils rich in saturated fatty acids (Augusto et al., 2012). Physical properties as

440

the melting point and crystallization patterns of oils and fats are also driven by the TAG

441

molecular species and their stereospecificity, while the position (sn-1, sn-2, or sn-3) of the

442

fatty acids on the backbone can influence quality properties such as “mouth feel” in fat-

443

containing foods (Karupaiah and Sundram, 2007).

444 445

Conclusions

446

High oil yield from pracaxi seeds was obtained with supercritical carbon dioxide at

447

mild conditions of temperature and short periods of time. Pracaxi oil was predominantly

448

composed of unsaturated fatty acids, mainly oleic and linoleic acids, and represents the

449

highest natural source of behenic acid ever reported. The TAG profile of PSO was mainly

450

composed of OOO, OOBe, and OOLi. The high stability to accelerated oxidation of the

451

pracaxi oil can be associated with its chemical composition (monounsaturated and saturated

452

fatty acids and phenolic compounds). Thermal analysis showed that pracaxi oil has a

453

typical behavior for vegetable oils and might present fat crystals at refrigeration

454

temperature, but is liquid at 20 °C. Although slight differences on the PSO quality

455

parameters obtained by SC-CO2 and by hexane were observed, the former has more

456

advantages as the product is solvent-free and the resulting cake may also be used for the

457

recovery of other compounds and nutrients, such phenolic compounds and proteins, or even

458

in biorefinery processes. Because of this composition, pracaxi seed oil can be considered a

459

specialty oil. These results suggest the application of PSO in different products such as

460

cosmetics, pharmaceutical, and food formulations, including frying oil.

20

461 462

Acknowledgments

463

The authors thank CAPES - Brazil for the postdoctoral scholarship granted to G.

464

L. Teixeira (processes n. 1795263 and 88882.316463/2019-01), and Amazon Oil company

465

for kindly donating the seeds of pracaxi. Thanks are also due to the Phytopathology

466

Laboratory (Labfitop/UFSC) for assisting in the spectrophotometric analysis. We are also

467

grateful to Ms. Rafaela Cristina Turola Barbi, Andrea Briones Gonçalves Bonassoli, and

468

Professor Dr. Rosemary Hoffmann Ribani for helping with the DSC analysis at the Federal

469

University of Paraná (Brazil). C. B. Gonçalves, J. M. Block, and S. R. S. Ferreira thank

470

CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the financial

471

support (processes n. 308615/2016-6, 310517/2015-0, and 404347/2016-9, respectively).

472 473

Declarations of interest: none

474

Funding: This work was supported by the Coordination for the Improvement of Higher

475

Education Personnel – CAPES, Brazil (grant numbers 1795263 and 88882.316463/2019-

476

01).

477 478

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638

25

Figure Captions

Fig. 1. Occurrence of pracaxi (Pentaclethra macroloba) in the South and Central America (a), the site where the seeds used in this study were collected (b), pracaxi seeds (c) and its main physical properties (in mm): height (d), width (e), and thickness (f). Maps by Leaflet and OpenStreetMap® retrieved from the Encyclopedia of Life (eol.org). Fig. 2. Extraction curve of pracaxi seed oil obtained using supercritical carbon dioxide at 250 bar, 40 °C, and 0.7 kg/h (a), response surface for the oil yield (b), and Pareto chart for the standardized effects of pressure and temperature on the oil yield (c). Fig. 3. Total phenolic compounds (TPC) and antioxidant activity of pracaxi oil extracts measured by different methods. Results are means ± SD (n = 3). GAE, gallic acid equivalents; AAE, ascorbic acid equivalents; FRAP, ferric reducing antioxidant power; CUPRAC, cupric ion reducing antioxidant capacity; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; ILP, inhibition of lipid peroxidation. Lowercase letters mean significant differences (p < 0.05) between samples by the Duncan test. Fig. 4. DSC thermograms showing the crystallization (a) and melting (b) behavior of pracaxi seed oils extracted by SFE using CO2 (P1-P5) and Soxhlet with hexane (PS). Fig. 5. Solid Fat Content of pracaxi seed oils obtained by SC-CO2 (P1-P5) and hexane (PS) estimated from the DSC melting curve.

26

Table Captions

Table 1. Experimental conditions and oil yield obtained from SFE of pracaxi seeds compared to the conventional Soxhlet method. Table 2. Physical and physicochemical characteristics of pracaxi seeds. Table 3. Fatty acid profile of pracaxi seed oil extracted by supercritical carbon dioxide (SC-CO2) or hexane (Soxhlet). Table 4. Triacylglycerol composition of pracaxi seed oils extracted by SC-CO2 at 40 °C and 300 bar and Soxhlet with hexane.

27

Table 1. Experimental conditions and oil yield obtained from the SFE of pracaxi seeds compared to the conventional Soxhlet method.

Sample

extraction

T (°C)

P (bar)

Density of 3 *

CO2 (kg/cm )

Viscosity of CO2 (µPa s) *

Time of

Oil yield (g/100 g)

extraction (min)

**

P1

40

200

839.81

78.32

20.77

P2

60

200

723.68

60.04

12.07

40

300

909.89

93.83

P4

60

300

829.71

78.82

41.26

P5

50

250

834.19

77.43

33.50 ± 3.39 ***

~ 68 (boiling point)

-

-

-

P3

PS *

Method of

Supercritical CO2

Soxhlet (hexane)

240

360

42.05

53.42 ± 1.75 ***

Source: NIST Chemistry WebBook (https://webbook.nist.gov);

**

Mass of the extract by the mass of dried material fed × 100;

***

Average of triplicate runs; P5 is the center point of the SFE.

28

Table 2. Physical and physicochemical characteristics of pracaxi seeds. Parameter

Mean ± SD

Weight (g)

6.36 ± 1.07

100-seed weight (g)

635.54 ± 1.07

Width (mm)

31.19 ± 3.01

Length (mm)

40.25 ± 9.60

Thickness (mm)

9.60 ± 1.44

Moisture (g/100 g)

4.02 ± 0.06

Proteins (g/100 g)

15.50 ± 0.01

Lipids (g/100 g)

53.42 ± 1.75

Ashes (g/100 g)

1.90 ± 0.04

Carbohydrates* (g/100 g)

25.17 ± 1.14

L

50.89 ± 0.86

a

8.81 ± 0.18

b

24.27 ± 0.78

C

101.91 ± 3.91

h

70.04 ± 0.27

*

Calculated by difference. Color parameters obtained from the ground pracaxi seed flour.

29

Table 3. Fatty acid profile of pracaxi seed oil extracted by supercritical carbon dioxide (SC-CO2) or hexane (Soxhlet). SC-CO2

Hexane

(40 °C/300 bar)

(Soxhlet)

nd

0.19

Palmitic (C16:0)

1.53

1.44

Stearic (C18:0)

2.68

2.54

Oleic (C18:1n9c)

53.46

53.27

Linoleic (C18:2n6c)

12.22

12.16

Linolenic (C18:3n3c)

0.11

0.11

Behenic (C22:0)

16.54

16.47

Lignoceric (C24:0)

11.13

11.57

Not identified

2.31

2.24

SFA

33.32

33.62

MUFA

54.33

54.10

PUFA

12.33

12.27

0.22

0.22

Fatty acid (%) Caproic (C6:0)

PUFA/MUFA

Results are the % distribution of fatty acids; nd: not detected.

30

Table 4. Triacylglycerol composition of pracaxi seed oils extracted by SC-CO2 at 40 °C and 300 bar and Soxhlet with hexane. Mass (wt%) SC-CO2 Hexane C68:0 BeBeLg 0.73 0.75 C56:1 POBe 0.95 0.90 C58:1 SOBe 2.17 2.07 C60:1 SOLg 1.00 0.98 C62:1 BeBeO 4.24 4.21 C64:1 LgOBe 5.42 5.61 C66:1 LgLgO 1.73 1.87 C52:2 OOP 1.79 1.68 C54:2 OOS 2.88 2.72 C58:2 OOBe 16.10 15.98 C60:2 OOLg 10.21 10.59 C62:2 BeBeLi 0.97 0.97 C64:2 LgLiBe 1.24 1.29 C52:3 PLiO 0.80 0.76 C54:3 OOO 20.24 20.07 C58:3 OLiBe 7.17 7.12 C60:3 OLiLg 4.59 4.76 C54:4 OOLi 13.21 13.12 C58:4 LiLiBe 0.89 0.88 C60:4 LiLiLg 0.57 0.59 C54:5 LiLiO 3.12 3.10 x: carbon number; y: double bonds. Fatty acids: P, Palmitic; S, Stearic; O, Oleic; Li, Group (x:y)

TAG*

Linoleic; Be, Behenic; Lg, Lignoceric.

31

Fig. 1.

Fig. 2. (a) 45

Oil yield (g/100 g)

40 35 30 25 20 15

250 bar 40 ºC

10 5 0 0

50

100

150

200

250

300

350

Time (min)

(b)

(c)

7.45576

P (bar) T (°C) -1.40097 TP 1.16892 p=0.05

400

(mgGAE/kgoil)

Fig. 3.

64

ab

48

ab

b c

c

32 16 0

a

(µM Fe2+/kgoil)

TPC

a

a

FRAP

2.1

b c

1.4

c

d

0.7 0.0

(% inhibition)

(mgAAE/kgoil)

a

CUPRAC

a

270

c 180

d

b

d

90 0

a 39

DPPH

a c

26

b

bc

d

13

(% inhibition)

0 29

ILP

a

a b

23 17 11

c

b

c

6 0 P1

P2

P3

P4

Samples

P5

PS

Fig. 4

Peak 1

Heat flow (mW)

a

P1 P2 P3 P4 P5 PS

Peak 2

Endo down

Cooling

-80 -70 -60 -50 -40 -30 -20 -10

0

10 20 30 40

Temperature (°C)

Heat flow (mW)

b

Shoulder P1 P2 P3 P4 P5 PS

Peak

Endo down

Heating

-80 -70 -60 -50 -40 -30 -20 -10

0

Temperature (°C)

10 20 30 40

Fig. 5.

P1 P2 P3 P4 P5 PS

Solid Fat Content (%)

100 80 60 40 20 0 -40

-30

-20

-10

0

10

Temperature (°C)

20

30

Highlights

- Supercritical fluid extraction of pracaxi seed oil using carbon dioxide is presented. - The highest content of oil was obtained by supercritical CO2 at 40 °C and 300 bar. - Oleic, behenic, and linoleic acids are the main fatty acids in pracaxi oil. - The triacylglycerol profile of pracaxi oil is mostly composed of Oleic-Oleic-Oleic. - Pracaxi oil extracted at 200 bar showed the highest phenolics content.