Oil extraction from macauba pulp using compressed propane

Oil extraction from macauba pulp using compressed propane

Accepted Manuscript Title: Oil extraction from macauba pulp using compressed propane Author: Caroline Portilho Trentini K´atia Andressa Santos Edson A...

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Accepted Manuscript Title: Oil extraction from macauba pulp using compressed propane Author: Caroline Portilho Trentini K´atia Andressa Santos Edson Antonio da Silva Vitor Augusto dos Santos Garcia Lucio Cardozo-Filho Camila da Silva PII: DOI: Reference:

S0896-8446(17)30031-1 http://dx.doi.org/doi:10.1016/j.supflu.2017.02.018 SUPFLU 3862

To appear in:

J. of Supercritical Fluids

Received date: Accepted date:

12-1-2017 23-2-2017

Please cite this article as: C.P. Trentini, K.A. Santos, E. Antonio da Silva, V.A.S. Garcia, L. Cardozo-Filho, C. da Silva, Oil extraction from macauba pulp using compressed propane, The Journal of Supercritical Fluids (2017), http://dx.doi.org/10.1016/j.supflu.2017.02.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Oil extraction from macauba pulp using compressed propane

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Caroline Portilho Trentinia , Kátia Andressa Santosb, Edson Antonio da Silva b,

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Vitor Augusto dos Santos Garciad, Lucio Cardozo-Filho c,d, Camila da Silva a,e

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a

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5790, 87020-900 Maringá-PR, Brazil.

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b

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(UNIOESTE), Rua da Faculdade 645, 85903-000, Toledo, PR, , Brazil.

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Programa de Pós-Graduação em Bioenergia, Universidade Estadual de Maringá, Av. Colombo,

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Centro de Engenharias e Ciências Exatas, Universidade Estadual do Oeste do Paraná

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Programa de Pós-graduação em Engenharia Química, Universidade Estadual de Maringá

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(UEM), Av. Colombo, 5790, 87020-900 Maringá-PR, Brazil.

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d

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(UNIFEOB), Av. Dr. Otávio Bastos, 2439, 13874-149, São João da Boa Vista - SP, Brazil.

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da Fonseca, 1800, 87506 -370, Umuarama, PR, Brazil.

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To whom correspondence should be addressed. Tel.: 55 44 3621 9300. Fax: (55) 44 3621 9326. E-mail: [email protected]

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ABSTRACT

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In this study, the extraction of macauba pulp oil (MPO) using compressed

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propane as a solvent was investigated, and compared with conventional (Soxhlet)

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extraction. Extractions with propane were carried out in order to investigate the

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effects of temperature (333-373 K) and pressure (4-12 MPa) on the oil yield and

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the chemical composition of the products. The effects of temperature and

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pressure on the yield were negative and positive, respectively, with a maximum

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yield of 23.08 wt% being obtained at 333 K and 12 MPa. The use of propane

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Departamento de Agronomia, Centro Universitário da Fundação de Ensino Octávio Bastos

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Departamento de Tecnologia, Universidade Estadual de Maringá (UEM), Av. Angelo Moreira

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allowed a fast extraction with a yield of ~86% in the conventional extraction. The

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fatty acids composition showed a predominance (~85%) of oleic and palmitic

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acids. The β-carotene and flavonoid contents were affected by the compressed-

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solvent extraction conditions and extraction method. Oil obtained using

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compressed-solvent extraction showed higher levels of phytosteroids and

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tocopherols and, consequently, a longer oxidation induction time.

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Keywords: Acrocomia aculeata oil, extraction, compressed propane, oil

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characterization.

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1. Introduction

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Macauba (Acrocomia aculeata) oil is an important resource with a productivity of 4 to 6 tons of oil per hectare [1].

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significant part of the plant is the fruit, because it has ~30% of oil in its

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composition [2,3]. However, macauba oil can also be extracted from the pulp and

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amond, and pulp has oil contents of 18.70% to 32.76% [4-8].

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The oil extracted from the pulp has an orange-yellow color, with a high

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concentration of oleic and palmitic acids [7,9-11]. Macauba pulp oil has active

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compounds in its composition, such as tocopherols [4,12,13], phytosterols [8,11]

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and β-carotene and flavonoids [5,14,8,15].

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As an alternative to the conventional methods of extraction using solvent

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and pressing, the extraction of vegetable oils using pressurized fluids under sub-

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or supercritical conditions has been reported. This extraction technique is

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flexible, due to the possibility of continuous modulation of the solvent power to

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adjust the selectivity of the supercritical fluid [16]. Also, the high diffusivity of

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the fluid ensures rapid extraction and the oil extracted with this technique is less

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subject to oxidation [17,18]. Additionally, the degradation of bioactive

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compounds is reduced or eliminated, resulting in a final product free of toxic

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solvent residues [19,20]. Carbon dioxide is generally used as the extraction solvent under

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supercritical conditions, but a limiting factor for its use is that it is associated

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with a low solubility of triacylglycerides [21]. Thus, relatively high pressures and

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longer extraction times are required to provide satisfactory yields [22-24].

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Propane (pressure and temperature critical of 369.67 K and 4.3 MPa,

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respectively), provides higher extraction yields than carbon dioxide, due to its

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high solvation power in relation to triacylglycerides at low pressures [21,22,25].

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This results in shorter extraction times and higher oil yields can be obtained with

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a smaller volume of solvent [22,26-28]. Additionally, it has been reported that

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propane is more efficient in the extraction of active compounds, such as

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phytosterols and tocopherols [16,29-33] and also carotenoids [31,34,35],

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compared to carbon dioxide, and provides extracts with high thermal stability

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[28,32].

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Publications in the literature report oil extraction from Acrocomia

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aculeata pulp using solvent extraction in a Soxhlet [3-5,7,9,12,36], pressing

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[7,37,38] and low-pressure extraction [8]. However, no previous studies on

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obtaining this oil using compressed propane as the solvent could be found.

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Recently, Nascimento et al. [24] reported the extraction of oil from macauba pulp

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(variety Acrocomia intumescens Drude) using supercritical carbon dioxide as the

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solvent. They obtained low oil yields with higher pressure (200 bar) and long

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extraction times (~200 min).

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In this context, the aim of this study was to obtain macauba pulp oil

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(MPO) using compressed propane as the solvent and investigate the effects of

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temperature and pressure in the oil yield. At the same time, extracts were

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obtained by the conventional Soxhlet technique, using n-hexane and

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dichloromethane as solvents, for comparative purposes. The extracts obtained

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applying the two techniques were characterized and compared in terms of their

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chemical compositions.

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2. Materials and methods

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2.1. Sample preparation

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Fruits of Acrocomia aculeata harvested in the Araripe Plateau (Global

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Positioning System coordinates 38°0' and 41°55' W; 70°10' and 7°50' S), Cariri

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region, Ceara State, Brazil were used in the experiments. The fruits were

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sanitized followed by the separation of the pulp, which was dried at 60 °C for 8 h

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(Marconi, MA035), obtaining a moisture content of 3.6±0.1 wt%. The dried

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material was milled in an electric mill (Marconi, MA 750) and classified using

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Tyler sieves (Bertel, ASTM). Particles with an average diameter of 0.5 mm were

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selected to conduct the experiments

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2.2 Reagents and standards

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Propane P.A. 99.5% (White Martins), n-hexane 99% (F. Maia) and

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dichloromethane 99.5% (Vetec) was used in the extractions. For β-carotene and

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flavonoid content, β-carotene standard >99.9% (Sigma-Aldrich), n-hexane (F

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Maia), ethanol 95% (Anidrol) and hydrochloric acid 37% (Nuclear) were used.

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For the determination of the tocopherol content, α, γand δ-tocopherol standards

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>99.9% (Sigma Aldrich), isopropanol (JT Baker, grau HPLC), methanol (JT

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Baker, grau HPLC) and ultrapure water (Milli-Q) were used. For determination

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of fatty acid composition and free glycerol compounds were used derivatizing

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agents BF3-methanol and N,O-bis (trimethylsilyl) trifluoro-acetamide-BSTFA

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with trimethylchlorosilane-TMCS, potassium hydroxide P.A. (Biotec), methanol

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PA (Vetec) and heptane 99.6% (F Maia), and internal standards of 5α-cholestane

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and methyl heptadecanoate >99.9% (obtained from Sigma Aldrich). Oxygen

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99.9% (White Martins) were used in DSC analysis.

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2.3. Oil Extraction

Propane was used as a solvent in the extraction, which was conducted with

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the experimental apparatus shown in Figure 1 and applying the procedure

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described in detail by Santos et al. [25]. The laboratory-scale unit consists of a

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gas cylinder, two thermostatic baths, syringe pumps (Teledyne ISCO 500 D), and

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a jacketed extraction vessel (1.95 cm of diameter and 19.4 cm of height) with a

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capacity of 58 cm3.

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To evaluate the effects of pressure and temperature, a 22 factorial design

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was adopted, with triplicates at the central point, where the pressure ranged from

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4 to 12 MPa and temperature from 333 K to 373 K. The pressure and temperature

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levels choice had as reference the phase behaviour of the pseudo-binary system

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was performed for the {Propane (1) + Moringa oleifera oil (2)} system [39]. The

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content of oleic acid (18:1) present in macauba pulp oil (MPO) and Moringa

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oleifera oil extracted with the pressurized propane are like, 76% and 61%,

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respectively. It is worth mentioning that the monounsaturated fatty acids have

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higher selectivity with compressed propane under low pressure. The solvent was

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pumped at a constant flow rate of 3 mL∙ min-1 and for each extraction the

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extraction vessel was loaded with ~17 g of pulp to form a bed of solids supported

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by two 200-mesh wire disks at both ends. The oil was collected in an amber glass vessel and its mass was determined

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at time intervals of 5 (0-30 min), 10 (30–60 min) and 20 (60–80 min) of

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extraction. The yields were calculated as the ratio of the extracted oil mass to the

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initial macauba pulp mass. The analysis of the experimental data, at the 95%

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confidence level, was performed using the Statistica 8.0 software program

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(STATSOFTTM, Inc.)

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The conventional oil extraction was performed in a Soxhlet apparatus, as

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recommended by Institute Adolfo Lutz [40], in order to compare the yield and

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characteristics of the oil with that obtained applying the compressed-solvent

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extraction technique. Dichloromethane and n-hexane were employed as solvents

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at their boiling points, for 480 min, and ~5 g of sample was used in each test.

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2.4. Oil Characterization

For the determination of the β-carotene content, samples were solubilized

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in n-hexane and a calibration curve was constructed from the dilution of a β-

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carotene standard to concentrations of 1.0 to 100 mg∙ L-1, which showed a

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regression coefficient of 0.998. The results were expressed in mg of β-carotene

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per 100 g of oil.

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The total flavonoid content was determined according to the procedure

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reported by Francis [41]. The oil (~1 g) was solubilized in 50 mL of ethanol/HCl

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(1.5 mol∙ L-1). The sample was homogenized, the solution was cooled and left to

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stand protected from the light for 12 h and then the absorbance was determined.

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The results were expressed in mg of flavonoids per 100 g of oil, using 76.6 as the

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flavonoids conversion factor. The β-carotene and flavonoids contents were determined based on the

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absorbance reading for the samples at 450 nm and 374 nm, respectively, using

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UV spectrophotometry (Femto, 700 plus).

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The α, γand δ -tocopherols were determined in a high performance liquid

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chromatograph (LC-20AT, coupled to a UV-VIS detector SPD-20A, Shimadzu)

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equipped with a C-18 column (Shim-pack CLC-ODS M, 25 cm x 4.6 mm, 5 μm).

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The method employed was that described by Freitas et al. [42] using methanol

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and ultrapure water as the mobile phase (96% of methanol and 4% of water).

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Samples were injected at 298 K with a loop of 50 μL, mobile phase flow rate of 1

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mL∙ min-1 and detection at 292 nm. Approximately 30 mg of the oil was

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solubilized in 1 mL of isopropanol, filtered through a nylon syringe filter

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(Analytical, 13 mm and 45 μm). To quantify the tocopherols in the samples,

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standard curves were constructed using chromatographic standards at

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concentrations of 0.5 to 5 mg∙ L-1 for αand γ,and 0.2 to 5 mg∙ L-1 for δ.

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The fatty acids composition and glycerol-free compounds were determined

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using a gas chromatograph coupled with a mass detector (Thermo-Finnigan),

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fitted with a capillary column Agilent HP-5MS (30 m x 0.25 mm x 0.25 µm),

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with the injection of 0.4 μL in split mode 1:10 and helium as the carrier gas at a

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flow rate of 1 mL∙ min-1. The identification of the components present in the

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samples was performed using the Xcalibur® software program (ThermoElectron).

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To determine the fatty acids composition, samples were derivatized

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following the methodology of AOAC Ce 2-66 [43] and analyzed applying the

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chromatographic conditions described by Trentini et al. [8], using methyl

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heptadecanoate as the internal standard. For the determination of free glycerol

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compounds, approximately 30 mg of oil was derivatized with 20 uL of

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BSTFA/TMCS with subsequent addition of 5α-cholestane and methyl

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heptadecanoate to quantify the phytosterols and free fatty acids, respectively. The

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solution remained for 30 min at 333 K. The analysis was performed applying the

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conditions reported by Santos et al. [25].

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The oxidative stability of the MPO was determined in a differential

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scanning calorimeter (DSC Shimadzu-60). In this analysis, 5 mg of oil was

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placed in an aluminum pan and then subjected to a flow of oxygen at 50 mL∙ min-

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413 K). The oxidation induction time (t0 ) was obtained from the oxidation curve,

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corresponding to the intersection of the baseline and the tangent line at the edge

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of the isotherm.

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. Each sample was submitted to four different temperatures (383, 393, 403 and

All of the analyses for the oil characterization were performed in

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duplicate, and the results are presented as mean values ± standard deviation. The

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data collected were subjected to ANOVA using Excel® 2010 and Tukey tests

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(with a 95% confidence interval) to evaluate differences between the results.

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3. Results and discussion

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3.1. Oil Extraction

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Table 1 shows the experimental conditions and results for the oil yield

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obtained in the conventional and compressed-solvent extraction of oil from

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macauba pulp. Analysis of the data obtained on the experimental conditions

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using propane, in the experimental range evaluated, indicates that the use of

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higher pressures at low temperatures favors the oil extraction (p<0.05). An

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increase in the temperature at constant pressure (compare runs 1 and 3) results in

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a decrease in the yield, due to a reduction in the solvent density. Under the

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conditions of run 1, the propane is in the gaseous state and its extractor power is

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lower compared with the other conditions evaluated.

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It can be seen from the data reported in Table 1 that the interaction

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between the temperature and pressure (experiments 2 to 5) had a moderate effect

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on the solvent density when compared to run 1, and similar yields were obtained

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(p>0.05).

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The extraction kinetics curves obtained with the use of compressed

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propane are shown in Figure 2, it was verified that extraction yield reached a

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plateau after a short extraction time (30 min for runs 2 to 5 and 20 min for run 1),

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which indicates the high solubility of the macauba pulp oil in this solvent. This

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behavior is one of the characteristics of the use of compressed propane as a

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solvent in the extraction of vegetable oils [39,44,45].

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Soxhlet extraction using n-hexane and dichloromethane as solvents

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resulted in similar yields (p>0.05) of 25.64% and 26.83%, respectively. The

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extraction with propane provided achieving ~86% of the yield obtained by

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Soxhlet. However, it is noteworthy that in this case the extraction time was 30

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min while for the conventional extraction it is 480 min, verifying that a high oil

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yield can be obtained with short extraction times using propane.

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In this study, the oil yields are higher than those reported by Hiane et al.

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[9] and Oliveira et al. [5] who used Soxhlet extraction with petroleum ether as

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the solvent and obtained oil yields of 19.3% and 18.70%, respectively. Recently,

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Rodrigues et al. [46] and Trentini et al. [47] reported obtaining 49.2% and

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44.78% of oil from macauba pulp for ultrasonic assisted extraction and

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pressurized liquid extraction, respectively, using ethanol as a solvent.

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3.2. Oil Characterization

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3.2.1 Fatty Acids

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The fatty acids compositions of the oils obtained are shown in Table 2.

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The macauba pulp oil contained higher concentrations of monounsaturated fatty

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acids, and the major fatty acids identified were oleic (20.93 to 29.75%) and

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palmitic (56.50 to 63.21%) acids. The polyunsaturated fatty acids identified were

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linoleic and linolenic acids.

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The statistical analysis showed a significant difference between the

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different solvents used in relation to the fatty acids composition, particularly the

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amount of oleic and palmitic acids extracted. The extraction of fatty acids may be

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related to the polarity of the solvents used, and long-chain fatty acids with at least

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one double bond, such as oleic acid, have lower polarity compared to other fatty

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acids. This characteristic explains the higher yield in the extraction of this

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compound when using low polarity solvents [48]. As can be observed in the

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extraction with dichloromethane, a solvent with higher polarity, less oleic acid is

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extracted, while with the use of solvents of lower polarity (like propane and n-

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hexane) the yields are greater. In the case of medium-chain fatty acids, such as

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palmitic acid, the solvents with high polarity demonstrate greater extraction

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efficiency.

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In studies reported by Jesus et al. [22], Pessoa et al. [27] and Silva et al.

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[4] on the extraction of oils from palm, pequi and Mucuna aterrima using

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propane, the different conditions applied in the extraction did not influence the

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fatty acids composition of the oils. The fatty acids composition of the macauba pulp oil obtained in the study

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reported herein is similar to others reported in the literature, for instance, Lescano

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et al. [7], Trentini et al. [8], Coimbra and Jorge [36], Navarro-Díaz et al. [38],

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Rodrigues et al. [46] and Trentini et al. [47]. In these studies, the levels of oleic

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and palmitic acid varied from 52.6 to 70.28% and from 17.65 to 27.39%,

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respectively.

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The contents of free fatty acids (FFA) and phytosterols (PHY) in the MPOs

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obtained are shown in Table 3. The FFA content of the oils ranged from 0.493 to

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1.66%, and the lowest levels were observed in the extractions with n-hexane and

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dichloromethane.

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Other authors have reported FFA contents of 0.83 to 9.43% for macauba

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pulp oil [5,9,36,49]. The differences observed may be associated with the region

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in which the fruits were harvested, climatic conditions, the time and temperature

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of the pulp drying before the oil extraction, and even the extraction method used,

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which can accelerate the formation of these compounds [3,36].

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As can be seen in Table 3, the phytosterols present in the macauba pulp oil

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were campesterol, estigmasterol and β-sitosterol. The total content of

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phytosterols was influenced by the experimental conditions, where the

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application of lower pressure and temperature favors the extraction of these

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compounds (p<0.05). Thus, 188.17 mg of PHY per 100 g of oil were obtained at

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333 K and 4 MPa.

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An increase in temperature causes a decrease in the efficiency of

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phytosterol extraction, which can be attributed to an increase in the vapor

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pressure of the active compounds in the oil, negatively influencing the extraction

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of phytosterols [50]. All of the extractions performed with propane led to a higher content of

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total phytosterols (p<0.05) when compared with the Soxhlet extraction. The

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average amounts of each phytosterol in the macauba pulp oil obtained in this

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study were 18.5, 7.9 and 73.6% for campesterol, stigmasterol and β-sitosterol,

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respectively, the best conditions providing 188.17 mg of PHY per 100 g of MPO.

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This content is comparable with recent reports for other vegetable oils obtained

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from the extraction with compressed propane, such as oils from crambe [25],

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perilla [28], flaxseed [51] and sacha inchi [20], with values of 201.05, 101.92,

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146.98 and 177.68 mg of PHY per 100 g of oil, respectively, being reported.

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Specifically for macauba pulp oil, Trentini et al. [8] reported 104.15 mg of PHY

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per 100 g of oil, applying extraction at low pressure using isopropanol as the

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solvent.

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3.2.3. Tocopherols

The results for the quantification of the tocopherols (α,γand δ) present in

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the macauba pulp oil obtained applying different extraction methods are

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presented in Table 4. The major tocopherol obtained in all extractions was γ -

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tocopherol, which is consistent with studies by Costa et al. [52], Przygoda and

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Wejnerowska [53] and Santos et al. [25] for oils extracted from buriti pulp,

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quinoa and crambe seeds, respectively.

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In the extractions carried out with propane under the conditions of higher

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temperature and lower pressure (333 K and 4 MPa), associated with a lower

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propane density, provided a higher concentration of total tocopherols (p<0.05).

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For the other conditions applied (runs 2 to 5 of Table 1), alterations in the

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temperature and pressure did not influence the solvent density and the extraction

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of these compounds (p>0.05). Silva et al. [28] evaluated the tocopherols content

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in perilla oil extracted using compressed propane and reported similar values

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under the conditions investigated, which were associated with a moderate

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variation in the solvent density, as observed in this study.

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In comparison with the concentration of tocopherols obtained with

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propane and in extractions using n-hexane and dichloromethane, oils obtained by

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Soxhlet extraction provided lower tocopherol contents, as also observed in

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studies by Zanqui et al. [51], Silva et al. [28] and Silva et al. [54] on oil

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extraction from flaxseed, perilla and pinhão endosperm, respectively. According

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to Nimet et al. [55] the lower concentration of tocopherols in oils obtained by

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Soxhlet extraction may be related to the long extraction time allowing the

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degradation of these compounds.

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3.2.4. β-carotene and flavonoids

The β-carotene and flavonoids content in the extracts obtained by Soxhlet

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extraction and compressed-solvent extraction are shown in Table 5. Higher

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concentrations of β-carotene and flavonoids were obtained from the extraction

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with propane, which again can be attributed to the short extraction time, the long

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Soxhlet extraction time allowing the degradation of these compounds [56,57].

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Furthermore, it was shown that propane is an excellent solvent for the extraction

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of β-carotene [22,31]. With the use of propane as a solvent, the extraction of β-carotene was

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favored by increasing the pressure and reducing the extraction temperature

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(p<0.05). Thus, the oil obtained at a higher solvent density, 333 K and 12 MPa,

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contained 356.05 mg of β-carotene per 100 g of oil. Trentini et al. [47] reported

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for pressurized liquid extraction with ethanol as solvent, obtaining 232.44 mg of

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β-carotene per 100 g of oil.

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As reported by Lu et al. [58], increasing the temperature at constant

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pressure causes a reduction in the solvent density, reducing its solvating power.

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Mustapa et al. [59] noted that the maximum solubility of β-carotene can be

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achieved using conditions where the solvent has a higher density, allowing the

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solvent to penetrate the vegetable matrix and dissolve this compound, which is

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located deep within the solid particles. The increased solubility of β-carotene in

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propane with the use of higher pressure is also reported elsewhere in the

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literature [34,35].

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In relation to the flavonoid contents, these varied from 11.03 to 13.12 mg

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per 100 g of oil, and a higher content of these compounds was favored by the

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application of low temperature and high pressure (333 K and 12 MPa). The oil

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extracted with propane contained higher contents of flavonoids, followed by the

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oil samples obtained using n-hexane and dichloromethane. Flavonoids vary in

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polarity and less polar solvents are efficient in the extraction of flavonoid

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aglycones [56].

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3.2.5. Thermal stability

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The thermal stability was evaluated using the oil samples extracted by the

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Soxhlet method, using n-hexane and dichloromethane, and the oil sample which

349

contained the highest amount of total tocopherols obtained with compressed

350

propane (333 K, 12 MPa). Table 6 shows the oxidation induction times (t0) as

351

well as the adjusted equations for the relationship between T and t 0, proposed by

352

Tan et al. [60], and their determination coefficients (R2), obtained for the samples

353

analyzed. The oxidation induction time (t0) was obtained from the oxidation

354

curve considering the intersection of the baseline and the tangent line at the edge

355

of the isotherm (Supplementary Material).

us

cr

ip t

347

The MPO extracted with propane showed a longer oxidation induction

357

time (120.68 min at 383 K) compared with the Soxhlet extraction. This result is a

358

characteristic of oils obtained by extraction using compressed propane, as

359

evidenced in studies by Santos et al. [25], Zanqui et al. [51] and Zanqui et al.

360

[18]. These authors reported oxidation induction times, at 333 K, of 420.2, 49.4

361

and 89.5 for crambe, flaxseed and chia oils, respectively.

363 364

M

d

Ac ce pt e

362

an

356

4. CONCLUSIONS

Macauba pulp oil (MPO) was obtained using compressed-solvent and

365

conventional (Soxhlet) extraction techniques. With the use of compressed

366

propane the best results were obtained by applying high pressure and low

367

temperature (12 MPa and 333 K), with a yield of 23.08 wt% being obtained

368

under these conditions. The extraction of MPO with subcritical propane provides

369

satisfactory extraction yields within 30 min, which represents ~86% of the yield

370

obtained applying conventional extraction. Palmitic and oleic acids were

371

predominant in the fatty acids composition of the oils obtained applying the

Page 15 of 37

different methods and conditions. The free fatty acids content was low (~2%),

373

and β-sitosterol and γ-tocopherol were the predominant phytosterol and

374

tocopherol in the MPOs, respectively. The compressed-solvent extraction favored

375

the extraction of β-carotene and flavonoids. The oil extracted with subcritical

376

propane had the longest oxidation induction time.

ip t

372

377 5. REFERENCES

cr

378 379

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632 633 634 635

d

631

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M

629

Page 26 of 37

635

Table 1. Experimental conditions and results for MPO extraction yields obtained

636

by compressed-solvent and conventional (Soxhlet) extraction techniques. P

Tim

T

(

ρ1

(

M

(g∙

(mi

K

P

cm-

n)

a

3

e

R

n )

Yield

(wt%)

)

Propane

us

) 1

3

80

Propane

7

1

3

2

3

M

3

0.11

9.77a

80

22.69b

0.41

d

3

Propane

4

Ac ce pt e

2

an

7 3

ip t

Solvent

cr

u

Oil

80

3 3

4

5

Propane

Propane

4

3 3

1

3

2

22.86b

0.44 80

23.08bA

0.47

3

80 22.8b±

5 3

8

3 6

n-hexane

0.232

0.42 480

25.64B ±

4

-

0.592

Page 27 of 37

2 3

480

26.83C ±

Dichloromet 7

1

0.102

hane 3

-

1

propane density; 2 Average value for three replicate runs ± standard deviation.

638

Means followed by the same lower case letters (comparison of runs with compressed propane)

639

and uppercase letters (comparison of conventional and compressed extractions) indicates no

640

significant difference (p>0.05).

Ac ce pt e

d

M

an

us

cr

ip t

637

Page 28 of 37

ip t cr

us

Table 2. Fatty acids composition of MPO obtained with compressed propane, n-hexane (HEX) and dichloromethane (DIC) as solvents. Compressed propane

acids

373 K 4 MPa

12:0

14:0

16:0

0.28±0

0.25±0.

.02a

08a

0.10±0

0.10±0.

a

MPa

353 K

7a

01a

02a

0.07±0.0

0.08±0.

0.05±0.

a

0.12±0.

0.08±0.0 a

HEX

DIC

-

-

8 MPa 0.30±0.

0.13±0

a

12

0.30±0.

02

a

333 K

0.22±0.0

.05

a

0.10± a

01

0

0.01

0.09±0.

0.09±0.

0.26±

a

a

a

.00

02

2

00

00

0.03

0.21±0

0.26±0.

0.24±0.0

0.27±0.

0.31±0.

0.02±

a

.02

27.43± ac

a

06

26.72± a

a

4

28.12±0. a

a

a

0

07

27.87± ac

29.16±1 a

b

0.00

20.93 b

2.28±0.

.09

26

5

09

65

0.10

60.85±

61.12±

61.16±0.

60.46±

59.33±1

63.21

0.33a

0.32a

40a

0.42a

.87a

±0.05b

1.89±0

18:1n-9

4 MPa

a

0.33

18:0

a

ce pt

10:0

12 MPa

333 K

Ac

8:0

373 K

ed

1

M an

Fatty

a

0.08

68

1.49±0.2

a

a

0.35

1.31±0. a

.45

±0.15

2.56±0.

2.08±

a

a

0.05a 0.15a 0.24a 29.75c 1.66a 56.50c

Page 29 of 37

2.93±0 18:3n-3

ab

.02

3.02±0.1

29.73

UFA3

69.96

70.27

MUFA4

60.85

61.12

9.11

9.15

-1

2

3

5

a

8.47± b

17

43

0.31

3.02±0.

2.78±0.

1.32±

a

b

15

c

8.55c 3.19d

13

0.07

29.92

32.47

23.39

31.85

69.81

70.08

67.53

73.00

68.24

61.16

60.46

59.33

63.21

56.50

8.20

9.79

11.74

8.65

ed

1

2.86±0.

30.04

5.42±0.

ac

9

SFA2

PUFA5

30.18

a

16

05

6.60±0.

a

ac

a

cr

ip t .08

5.63±0.3

us

ac

6.29±0.

M an

6.18±0

18:2n-6

9.62

4

5

Results in g 100 g of oil; Saturated Fatty Acids; Unsaturated Fatty Acids; Monounsaturated Fatty Acids; Polyunsaturated Fatty Acids.

Ac

ce pt

Means followed by the same letter (in each row) indicate no significant difference (p>0.05).

Page 30 of 37

Table 3. Free fatty acids (FFA) and phytosterol (PHY) in MPOs obtained with compressed propane, n-hexane (HEX) and dichloromethane (DIC) as solvents. FFA

Phytosterol (mg of PHY per 100 g of oil)

Content Campesterol

Stigmasterol

β-sitosterol

33.87±3.70

13.77±2.03

132.97±0.58

25.88±1.43

13.13±0.19

Pa

4 Pa 3

Pa 3

1.30±0.02 b

M

Ac ce pt e

3

180.61±2.25 a

121.59±1.91

160.60±0.66b

d

Pa

an

1.40±0.05 b

us

1.66±0.03aA

4

3

cr

mpressed propane 3

Total

ip t

(%)

26.75±0.84

11.76±1.60

149.65±1.59

188.17±0.83 cA

30.67±2.13

15.26±0.81

125.94±0.73

171.87±2.05d

31.31±1.15

12.05±0.17

130.45±1.40

173.81±0.43d

1.34±0.01 b

1.34±0.10 b

8 Pa nventional (Soxhlet) extraction

Page 31 of 37

X

0.493±0.01B

2.84±0.18

3.24±0.66

17.31±2.78

23.39±1.94 B

IC

0.524±0.07B

6.71±1.07

4.81±1.72

35.34±1.84

46.86±0.95 C

Means followed by the same lower case letters (comparison of runs with compressed propane) and uppercase letters (comparison of conventional and compressed extractions) indicate no significant difference

ip t

(p>0.05).

propane, n-hexane (HEX) and dichloromethane (DIC) as solvents.

γ

δ

Compressed propane 373 1.92±0.02

14.57±0.30

12 MPa 333 K; 4 MPa

Ac ce pt e

373 K;

18.66±0.31 aA

d

MPa

Total

2.17±0.02

M

K; 4

an

α

us

Tocopherol (mg per 100 g oil)

cr

Table 4. Tocopherols (α, γand δ ) content in MPOs obtained with compressed

2.02±0.01

10.42±0.10

1.84±0.01

14.28±0.10b

1.71±0.02

9.97±0.02

2.47±0.01

14.15±0.02b

1.84±0.01

10.06±0.16

2.05±0.01

13.95±0.16b

333 K; 12 MPa

Page 32 of 37

353 K; 8

1.85±0.05

10.72±0.13

1.74±0.03

14.31±0.21b

MPa Conventional extraction (Soxhlet) 2.36±0.01

10.13±0.19

0.67±0.03

13.16±0.18 B

DIC

1.73±0.03

10.24±0.21

0.72±0.02

12.69±0.21 B

ip t

HEX

cr

Means followed by the same lower case letters (comparison of runs with compressed propane) and uppercase letters (comparison of conventional and compressed extractions) indicate no significant

an

us

difference (p>0.05).

Table 5. β-carotene and total flavonoid contents in MPOs obtained with compressed

β-carotene

M

propane, n-hexane (HEX) and dichloromethane (DIC) as solvents.

Ac ce pt e

d

(mg per 100g of

Flavonoid

(mg per 100g

oil)

of oil)

373 K; 4 MPa

136.66±1.65 a

11.03±0.07 a

373 K; 12 MPa

185.03±1.55b

12.34±0.10 b

333 K; 4 MPa

303.70±1.89 c

12.21±0.04 b

333 K; 12 MPa

356.05±1.67dA

13.12±0.10cA

353 K; 8 MPa

347.21±4.65d

11.06±0.06 a

Compressed propane

Conventional extraction (Soxhlet) HEX

213.33±1.97 B

12.32±0.07 B

Page 33 of 37

282.41±1.57 C

DIC

11.36±0.04C

Means followed by the same lower case letters (comparison of runs with compressed propane) and uppercase letters (comparison of conventional and compressed extractions) indicate no significant difference (p>0.05).

ip t

Table 6. Oxidation induction times obtained by DSC analysis for MPOs extracted with propane, n-hexane (HEX) and dichloromethane (DIC).

3

4

4

8

9

0

1

3

3

3

3

K

K

K

K

5

2

us

3

cr

(min)

2

0

4

Ac ce pt e

0

Propane

equation

9

2

d

2

R

M

1

Regression

an

Solvent

.

4

log10t 0

9

2

6

5

T=167.08-27.32*

.

.

5

.

.

9 5

1

8

8 4

1

2

9

0

8

.

.

HEX

.

.

T=157.75-29.24* -

8 7

9 log10t 0

8

9 4

0

0

6

2

9 9

DIC

T=153.29-

0

23.55*log10t0

.

5

8

.

Page 34 of 37

.

.

4

9

7

2

0

9

0

4

Ac ce pt e

d

M

an

us

cr

ip t

1

Page 35 of 37

Figure 1. Schematic diagram of the extraction unit used in this study: C-1 carbon dioxide and propane cylinder; CB-01 and CB-02 circulation baths; P syringe pump; E extractor; S sampling trap; V-1 ball valve; V-2 and V-3 needle valves; V-4 micro-

ip t

metering needle valve.

cr

Figure 2. Kinetics of the extraction of macauba pulp oil (MPO) using compressed

us

propane (▲353 K/8 MPa), (○373 K/12MPa), (●373K/4MPa), (□333K/12MPa),

Ac ce pt e

d

M

an

(◊333K/4MPa).

Page 36 of 37

Calibration curve of β-carotene in n-hexane (y=0.0169*x-0.0007; R2=0.9981). Calibration curve of α-tocopherol (y=0.0002*x-0.3636; R2 =0.9909). Calibration curve of γ-tocopherol (y=0.0003*x+0.1790; R2=0.9947). Calibration curve of δ-tocopherol (y=0.0001*x+0.1718; R 2=0.9914). Isothermal curves for macauba pulp oil extracted with propane subjected to

Ac ce pt e

d

M

an

us

cr

ip t

oxygen flow at 383 K.

Page 37 of 37