Journal Pre-proof

Extraction and assessment of oil and bioactive compounds from cashew

nut (Anacardium occidentale) using pressurized n-propane and ethanol
as cosolvent

Ana Beatriz Zanqui, Claudia Marques da Silva, Jéssica Barrionuevo

Ressutte, Damila Rodrigues de Morais, Jandyson Machado Santos,
Marcos Nogueira Eberlin, Lúcio Cardozo-Filho, Edson Antônio da
Silva, Sandra Terezinha Marques Gomes, Makoto Matsushita

PII: S0896-8446(19)30688-6
DOI: https://doi.org/10.1016/j.supflu.2019.104686
Reference: SUPFLU 104686

To appear in: The Journal of Supercritical Fluids

Received Date: 23 July 2019

Revised Date: 2 October 2019
Accepted Date: 15 November 2019

Please cite this article as: Zanqui AB, da Silva CM, Barrionuevo Ressutte J, de Morais DR,
Machado Santos J, Nogueira Eberlin M, Cardozo-Filho L, da Silva EA, Marques Gomes ST,
Matsushita M, Extraction and assessment of oil and bioactive compounds from cashew nut
(Anacardium occidentale) using pressurized n-propane and ethanol as cosolvent, The
Journal of Supercritical Fluids (2019), doi: https://doi.org/10.1016/j.supflu.2019.104686
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© 2019 Published by Elsevier.

 Extraction and assessment of oil and bioactive compounds from cashew nut

(Anacardium occidentale) using pressurized n-propane and ethanol as cosolvent

Ana Beatriz Zanquia*, Claudia Marques da Silvac, Jéssica Barrionuevo Ressutteb, Damila

Rodrigues de Moraisd, Jandyson Machado Santosd,e, Marcos Nogueira Eberlind, Lúcio

Cardozo-Filhoa*, Edson Antônio da Silvaf, Sandra Terezinha Marques Gomesc, Makoto

Matsushitac

of
a b
Departamento de Engenharia Química, Departamento de Ciência de Alimentos and
c

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Departamento de Química, Universidade Estadual de Maringá, UEM, Maringá, PR, 87020-

900, Brazil.
d -p
ThoMSon Mass Spectrometry Laboratory, Instituto de Química, Universidade de Campinas1

- UNICAMP, Campinas, SP, 13083-970, Brazil.

re
e
Departamento de Química, Universidade Federal Rural de Pernambuco, Recife, PE, 52171-
lP

900, Brazil.
f
Departamento de Engenharia Química, Universidade Estadual do Oeste do Paraná, Toledo,

85903-000, PR, Brazil.

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*Corresponding author. E-mail address: lucio.cardozo@gmail.com

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Graphical abstract
Highlights

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 The increase in temperature and ethanol favored the increase of extraction yield

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 The amount of oleic acid obtained was similar to that found in olive oil

 The amount of oil obtained ranged from 19.2 to 33.1%


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There was variation in the amount of sitosterol and tocopherols
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 The best conditions evaluated were 60°C and 6% ethanol as cosolvent
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Abstract: Cashew nuts in natura were crushed and subjected to lipid extraction using

pressurized propane. There was variation in the temperature (30-60°C) and percentage of the
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cosolvent ethanol (0-6%) using a 2² factorial design with central point in three replicates. The

lipid composition was determined and compared to the composition of the ethereal extract
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obtained by Soxhlet method (SE). The amount of oil extracted ranged from 19.2 to 33.1%. The

extraction performed under the conditions of 8 MPa, 60°C using 6% ethanol corresponded to
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83.7% of the total lipids. The higher the temperature and percentage of ethanol in the propane,

the higher the extraction yield. Thirteen fatty acids were quantified, among them, oleic and

linolenic acid were predominant. Tocopherols and sitosterol were higher in the oils obtained

using pressurized propane and ethanol. The mathematical model of Sovová was adjusted to the

kinetic curves of extraction and represented them satisfactorily.

Keywords: Fatty acids, food chemistry, bioactive compounds, mathematical modeling,

ethanol, pressurized fluid.

Abbreviations:

TL: total lipids; CPE: compressed n-propane extraction; mF: mass flow rate of the solvent (g

min-1); S: solubility of oil in the solvent (g oil g-1 solvent); t: extraction time (min); q0: initial

oil concentration in the solid matrix (g oil g-1 solid); ms: solid mass on an oil-free basis (g); KFa:

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solvent-phase mass transfer coefficient (min); KSa: solid-phase mass transfer coefficient (min);

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r: easily accessible oil mass; tCER: time at which the extraction of the oil from the inside of

particles starts (min); tFER: time at which the extraction of easily accessible solute ends (min);
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Z: dimensionless parameter of Sovová model; Y: dimensionless parameter of Sovová model;
exp
t0: induction time; mcalc
oil,j , calculated mass of the oil extracted (g); moil,j : mass of oil
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experimentally obtained (g); n-exp: the number of experimental data points on the kinetic
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curve.
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Greek letters

ρbed, bed density (g cm-3); ρF, solvent density (g cm-3); ρa, apparent density; ρt, true density (g
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cm-3); ε, bed porosity.

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1. Introduction

The nutritional benefits of oilseeds can be attributed to their lipid content, especially

unsaturated fatty acids (FA), phytosterols and tocopherols [1]. Studies have shown that humans

who have a diet that includes omega-3 fatty acids, polyunsaturated fatty acids (PUFA) and
oleic acid have a lower occurrence of cardiovascular diseases and cancer; besides that, the

consumption of oleic acid is directly linked to antithrombotic effect [2,3].

Native to the northeastern region of Brazil, the cashew nut (Anacardium occidentale) is

commonly consumed as snacks and used in recipes, furthermore, the cashew nut oil contains

large amounts of oleic acid and other compounds with bioactive activity. Its oil is also used in

cosmetic, pharmaceutical and food industries, therefore, it is necessary that the extraction

method preserve he oil quality be free of toxic residues [4,5].

There are some disadvantages to obtaining vegetable oils by conventional extraction methods,

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such as the toxicity of the solvents, long extraction time using high temperatures, and an

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evaporation step. The lipid extraction method used to obtain the oil may also interfere with the

results of bioactive compounds. In addition to traditional methods, a method that is attracting

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researchers’ interest is supercritical fluid extraction (SFE), which uses non-toxic solvents and

mild temperatures at high pressures to extract lipids of various oilseeds. The most commonly
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used solvent is carbon dioxide (CO2), however, since vegetable oils are sparingly soluble in
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carbon dioxide, the researchers are evaluating the effectiveness of n-propane in pressurized

extractions. Its use provides shorter extraction times than using CO2 due to its higher lipid

solubility. In addition, n-propane is non-toxic. Studies have shown good results with n-propane
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compressed fluid. In addition to manipulating temperature and pressure of the fluid, it is

interesting to insert a cosolvent, or a modifier such as ethanol, which is able to modify the
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mixture polarity and density, aiding in oil extraction and bioactive compounds [6,7].
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One way of observing the estimation of viability of the process on an industrial scale is to

evaluate the mass transfer process from the extraction kinetics, considering the steps described

in the overall extraction curve (OEC): constant extraction rate (CER) and falling extraction rate

(FER), described by the mathematical model proposed by Sovová [8].

Light hydrocarbons, such as n-propane, appear to be a promising solvent for extraction of the

edible oil. Because ethanol is a suitable solvent for the recovery of phytosterols, the mixture of

these solvents (ethanol and propane) can synergically combine to achieve the highest yields

and phytosterol content. In addition, the use of ethanol decreases the flammable aspects of n-

propane and increases green-solvent aspects [9]. Thus, the compressed n-propane extraction

(CPE) with ethanol as cosolvent should be better studied.

So, the aim of this study was to obtain oil from cashew nut using extraction with pressurized

n-propane and ethanol as cosolvent. For this purpose was evaluated the influence of

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temperature and addition of a cosolvent (ethanol) on the extraction of cashew nut oil in terms

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of total yield, concentration of fatty acids, tocopherol and phytosterol and triacylglycerols in

comparison with the ethereal extract obtained by Soxhlet extraction (SE), using mass
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spectrometry chromatography (GC-MS) and flame ionization detector chromatography (GC-

FID), as well as by direct analysis technique such as EASI-MS (easy ambient sonic-spray
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ionization mass spectrometry). Besides, the extraction kinetic curves were described by the
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Sovová model, applied to calculate mass transfers of the process.

2. Materials and methods

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2.1 Samples

Three kilograms of cashew nuts were obtained from farmers in Campo Grande - MS (Brazil).
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The in natura samples were manually peeled. They were shredded in a multiprocessor and
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separated using the portion that passed through a 12 mesh Tyler series sieve and did not pass

through a 14 mesh Tyler series sieve (WS Tyler, USA) (granulometry 1.18-1.40 mm). The

samples with 10.05% relative humidity were vacuum packed in polyethylene bags and frozen

at -18°C.
2.2 Conventional lipid extraction by Soxhlet

The Soxhlet extraction (SE) method was carried out using 3.00 g (± 0.1 mg) of sample as

proposed by Soxhlet [10], AOAC method 920.39, using 40.00 mL of a 1:1 (v/v) petroleum

ether/ethyl ether mixture refluxing at boiling point for 16h. The residual solvent of the oil was

evaporated in a heated water bath at 65°C and total lipid (TL) content was determined

gravimetrically by analytical scale.

2.3 Compressed n-propane extraction (CPE)

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The extractions with n-propane were carried out in an experimental apparatus previously

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described by Silva et al. [11]. The experiments were performed using pressure of 8 MPa, using

a 2² factorial design with central point in triplicate in the experimental range at temperature of
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30–60°C and proportion of ethanol of 0–6%, according to Table 1. The extraction conditions

were chosen based on experimental vapor-liquid equilibrium (VLE) data for the binary system
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propane-ethanol as described by Zabaloy et al. [9]. For the pressure of 8 MPa and range of
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temperature used, the solvent mixture propane-ethanol is above saturation line. The extractions

were performed using 15.0 g of sample per extraction, and porcelain beads were added to

complete the total volume of the extraction vessel (53.4 cm²). Propane (99.50% purity, Linde
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gas, São Paulo, Brazil) and ethanol (99.50% purity, Anidrol, São Paulo, Brazil) were

compressed through a syringe pump (model 500D, Teledyne Isco, USA) at 5°C. A flow rate of
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2 mL min-1 of solvent was used for 60 min, controlled by a micrometric valve (Parker
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Autoclave Engineers, Pennsylvania, USA) maintained at 80ºC, above the boiling temperature

of ethanol under ambient pressure (78.30°C) using a thermoregulator (Tholz, model CTM-

04E). The total lipids extracted were collected in pre-weighed glass flasks at four initial cycles

of 5 min and four cycles of 10 min (total of 60 min) and the extraction yield was determined
gravimetrically by analytical scale. The analyses were carried out in a completely randomized

order.

2.4 Apparent density and true density

Some parameters were calculated for the modeling process by Sovová [8]. The true density (ρt)

was determined by helium pycnometry using pycnometer Quantachrome Ultrapyc 1200e

(Quantachrome, Germany). The apparent density (ρa) of the particles was calculated using

equation 1.

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m sample

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𝜌a = (1)
vc

where: ρa = apparent particle density; m sample = mass of cashew nut introduced into the
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extraction column; and vc = volume of extraction column.
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2.5 Bed porosity
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Using Equation 2, Bed porosity (ε) was calculated:

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ρa
ε = 1- ρt (2)

where: ε = bed porosity; ρa = apparent particle density; and ρt = true density of the particles.
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2.6 Kinetics of the cashew nut oil extraction

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The second-order model of Sovová [8] was used to describe the behavior of the kinetic curves

of the extractions, considering that the process was isobaric and isothermal, that the particles

of the solid sample are composed of broken and unbroken cells, and that the distribution of the

solute in the bed and the particle size were homogeneous. In addition, the process considers

two mechanisms of mass transfer and three distinct periods of extraction: constant extraction
rate (CER), falling extraction rate (FER) and diffusion controlled (DC). The equations that

describe the Sovová model are as follows (3-10):

moil = mF St[1- exp(-Z)] for 0 ≤ t ≤ tCER (3)

moil = mF S[t-tCER exp(Zm(t)-Z)] for tCER ≤ t ≤ tFER (4)

𝑆 Wq0 mm
moil = mS {q0 - W ln [1+ (exp ( ) -1) exp (( m F ) (tCER -t)) r]} for t ≥ tFER (5)
S S

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ZS 1 W mF
Zm(t) = ln { [exp [( ) (t-tCER ] -r]} (6)

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Wq0 1-r mS

Z=
kF ams ρF
mF ρbed
-p (7)
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mS kS a
W= (8)
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mF (1-ε)

(1-r)mS q0
tCER =
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(9)
Sb ZmF
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m Wq0
tFER = tCER + WmS ln [r+(1-r)exp ( )] (10)
F S
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where: mF = mass flow rate of the solvent (g min-1); S = solubility of oil in the solvent (g oil g-
1
solvent); t = time (min); q0 = initial solute concentration in the solid in terms of mass of solute

per unit mass of insoluble solid (g oil g-1 solid); mS = solid mass on an oil-free basis (g); ρbed =

bed density (g cm-3); ρF = solvent density (g cm-3); KFa = solvent-phase mass transfer coefficient

(min-1); ε = bed porosity; KSa = solid-phase mass transfer coefficient (min-1); r = easily
accessible oil mass; tCER = time at which the extraction of the oil from the inside of particles

starts (min); and tFER = time at which the extraction of easily accessible solute (min) ends. The

dimensionless parameters of the Sovová model (Z, W, and r) were determined using the

downhill simplex method [12], by minimizing the following objective function (Equation 11):

n-exp exp
F = ∑j=1 (mcalc
oil,j - moil,j ) (11)

exp
where: mcalc
oil,j = calculated mass of the oil extracted (g); moil,j = mass of oil experimentally

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obtained (g); n-exp = number of experimental data points on the kinetic curve.

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2.7 Determination of fatty acids

The FA were methylated according to Hartman and Lago [13] using basic catalysis followed
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by acid catalysis. Fatty acid methyl esters were analyzed on Trace Ultra 3300 Thermo gas
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chromatograph equipped with a flame ionization detector and CP-7420 fused silica capillary

column (100 m long, 0.25 mm internal diameter and 0.25 μm cyanopropyl) according to Sargi
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et al. [14]. Fatty acids identifications were achieved by comparing retention time (RT) with

Sigma standards (USA), and the calculation of peak areas was done using the ChromQuest 5.0
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software. The FA quantification (in mg FA g−1 of TL) was determined in relation to the

standard of methyl tricosanoate (Sigma), and the calculations were done according to Joseph
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and Ackman [15] and Zara et al. [16].

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2.8 Triacylglycerol composition

The TAG profile of cashew nut oil was obtained using a EASI-MS on a quadrupole mass

spectrometer (LCMS-2010 EV, Shimadzu, Japan) as described previously by Zanqui et al. [17].

The main oil components were identified by specific values of m/z for TAG ions according to

Cabral et al. [18] and Haddad et al. [19], in the positive ion mode acquisition, EASI(+)-MS.
The EASI source was operated using methanol and nitrogen gas (N2) as nebulization gas with

flow rates of 20 μL min-1 and 2 L min-1, respectively. The surface-entrance angle of the mass

spectrometer relative to the EASI source was set at 30º. For direct analysis, one drop of cashew

nut oil (about 1 μL) was placed on Kraft paper, and data was collected for 30 s with scanning

over in a range of m/z 100-1200. The analyses were performed in triplicate and the data was

obtained and processed using Solution v.3.70 software (Shimadzu, Japan).

2.9 Analysis and quantification of phytosterols and tocopherols

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Phytosterols and tocopherols present in the oils were simultaneously analyzed by gas

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chromatography (GC) in Thermo Focus GC model (Thermo-Finnigan), coupled with a mass

spectrometer (MS) model DSQ II (Thermo-Finnigan) equipped with electron ionization source
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(EI), according to Du and Ahn [20]. The compounds were derivatized using N-O-

bis(trimethylsilyl)trifluoracetamide (BSTFA; Sigma-Aldrich Chemical Co., USA) according

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to Beveridge et al. [21]. The compounds were separated in a capillary column with 5% phenyl,
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95% methylpolysiloxane (DB-5) of fused silica with 30 m, 0.25 mm i.d., and 0.25 mm thick

film stationary phase (J & W Scientific, Folson, USA), using a flow rate of the carrier gas (He

- 5.0) of 1.0 mL min−1. The temperature of the injector, detector and the transfer line between
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the GC and MS was 280, 280, and 250°C, respectively. The analyses were performed in

triplicate, using 2μL for each injection, with 1:10 split rate.
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The compounds were identified using the data acquisition system by Xcalibur Software [22]
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that accompanies the database of spectra contained in the NIST MS Search version 2.0 spectral

library [23], and the quantification of the compounds was performed against the internal 5-α-

Cholestane standard (Sigma) according to Li et al. [24].

2.10 Statistical Analysis

The results were submitted to analysis of variance (ANOVA) at 5% significance, by Tukey test

using Statistica Software, version 8.0 [25]. The main effects and interactions were calculated

by Design-Expert 7.1.3 software [26], which evaluated the effect of independent variables on

response.

3. Results and Discussion

3.1 Extraction yield

The Soxhlet method is able to extract all lipid content from samples. Thus, the comparison of

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n-propane CPE with SE is effective to show the efficiency of the new technique employed. In

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addition, the literature has already shown the use of supercritical carbon dioxide in cashew nut

extraction [27].
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The pressure was fixed in order to observe the influence of temperature and the percentage of

ethanol in the solvent, as studies showed that the pressure does not bring significant variations
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in the extraction yield of other nuts [17,28].
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The particle size chosen was based on previous experiments and studies with other matrices

showing that the smaller the particle size, the better the extraction yield results [28].
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Experimental conditions and total yield for the extraction of oil from cashew nut by CPE and

SE methods are shown in Table 2. The higher extraction yield was obtained by SE method

(33.1%), using petroleum ether and ethyl ether; potentially because of the polarity of these
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solvents, other compounds were able to be extracted, thus exceeding estimated extraction yield
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[29].

The amount of oil extracted by CPE, with constant 8 MPa of pressure, improved with

increasing temperature and percentage of ethanol. This can be observed by comparing

experiments A and B, where the increase in temperature (30 to 60°C) increased extraction yield
from 19.2 to 22.3%, which also occurs in experiments C (30°C and 6% Et) and D (60°C and

6% Et). The addition of the cosolvent ethanol also favored the increase in the extraction yield

at the two temperatures evaluated; it was found up to 27.7% oil using 60°C and 6% ethanol in

the extraction of cashew nuts with pressurized propane. This result corresponds to 83.7% of

the ethereal extract obtained by SE method, free from residues of toxic solvents and in shorter

time. The mixture of ethanol and n-propane represents approximately 5% increase in extraction

yield.

For comparison to roasted nuts, the dry weight lipid percentage was calculated, shown in Table

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2. Lameira et al. [27] extracted toasted cashew nut lipids with granulometry of 0.50-0.85 mm

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using supercritical CO2 under various conditions, varying the temperature from 40 to 80°C and

the pressure from 10 to 17 MPa, and achieved yields of 4.5 to 32%, which are in agreement

with our data.

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These variations were described by the regression analysis (Table 3) which indicated a
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significant linear model for two-factor interaction (2FI) described by Equation 12, where T
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corresponds to the temperature variable and Et to percentage of ethanol in propane solvent.

𝑌𝑖𝑒𝑙𝑑 = 26.10 + 3.90 𝑇 + 5.35 𝐸𝑡 + 2.35 𝑇 𝐸𝑡 (12)

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The determination coefficient (R²), adjusted determination coefficient (R²adj) and the
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coefficient of variation (CV) for the model were 0.9999, 0.9997 and 0.40% respectively. The
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Model F-value of 6580.67 implies that the model is significant and values of Prob > F less than

0.05 indicate that the model terms are significant. The temperature and percentage of ethanol

showed a synergetic effect; the increase of these levels resulted in the highest quantities of oil

(Figure 1).
3.2 Mathematic modeling of the extraction

For the implementation of the Sovová mathematical model, the following characteristics were

required: initial oil concentration in the inert solid = 0.72 g-1 and bed porosity = 0.76. Table 4

shows the adjustable parameters for the Sovová model and the cashew nut oil solubility in the

solvent propane and propane with ethanol, which was determined using the dynamic method,

and was calculated from the slope of the kinetic curves. The model adjusted the kinetic curves

of extraction, as corroborated by the values of R² and Figure 2, where A (30°C/0% Et), B

(60°C/0% Et), C (30°C/6% Et), D (60°C/6% Et) and E (45°C/3% Et) indicates the experimental

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tests.

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The change in the slope of the kinetic extraction curves occurs due to the variations of the

convective and diffusive mass transfer mechanisms. The determination of the velocity of the
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mass transfer process occurs mainly due to the convection mechanism in the fluid phase, and

as the gradual removal of the oil occurs, a discontinuity effect occurs in the surface layer. From
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that moment, a decreasing rate of extraction can be observed, which initiates a diffusive
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process, during which the oil of difficult access starts to be extracted.

It can be observed that the lower solubility value of the oil in the solvent coincided with the

test with lower extraction yield (A – 30°C/0% Et).The values of mass transfer coefficients in
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the solvent phase (KFa) were higher than KSa (solid phase), indicating that easily accessed oil

provided higher extraction yield. The KSa values were lower than the KFa values, which
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indicates more difficulty in the diffusion process and consequently the solubilization of the oil
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of difficult access, as verified by other authors [11,30].

The tCER values, which indicate the time at which the extraction of the oil from the interior of

the particles starts to be extracted, were lower for the two extractions at higher temperatures

(60°C) for or about 28 min, indicating that higher temperatures favor the penetration of the
fluid inside the particles to extract the oil of difficult access. The tFER values show the time that

the extraction of the easily accessible oil would end, that is, the extraction can be extended up

to 145 min for better yields.

3.3 Fatty acids composition

Table 5 shows the 13 fatty acids that were quantified in cashew nut oils obtained by CPE (A-

E) and Soxhlet methods (F) and the sum of saturated (SFA), monounsaturated (MUFA) and

polyunsaturated fatty acids (PUFA). The fatty acids found in greater quantity were: oleic

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(18:1n-9), linoleic (18:2n-6), palmitic (16:0) and stearic (18:0) acids, as well as two fatty acids

belonging to the omega-3 series in small amounts (18:3n-6 and 18:3n-3).

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The predominant fatty acid was oleic acid, an omega 9, with an average of 635 mg of FA per

gram of lipid, showing no significant variations between extractions, which proves that the
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innovative method by CPE does not degrade the main fatty acid present in the oil.
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The majority fatty acids and sum of FA are shown in bar graphs in Figure S1 in the

supplemental material.
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Olive oil is often consumed for its functional properties, because it contains oleic acid in

percentages varying between 60 and 70% [31]. In our study, cashew nut oil showed percentages
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of oleic acid equal to those obtained in olive oil and higher than the amount of 18:1n-9 found

in canola, soybean, sunflower and sesame oils [32–34].

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Linoleic acid (18:2n-6), a precursor of eicosanoids that have anti-inflammatory functions in the

body, was quantified in all samples with values around 110 mg g-1 of FA, in accordance with
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values found by other authors [35].

Table 5 also shows that the composition of monounsaturated fatty acids (MUFA) in cashew

oils was two times higher than that obtained for sesame oil, three times higher than that found

in flaxseed oil and nine times higher than that found in chia oil, extracted by the methods of
SE, SFE and enzymatic extraction [7,33,36]. The ingestion of MUFA can aid in the prevention

of chronic and cardiovascular diseases, so eating foods containing these FA could be beneficial.

3.4 Triacylglycerol Composition (fingerprinting)

EASI-MS provides faster analyses with no sample preparation and has been widely applied for

vegetable oil typification and quality control [37]. EASI(+)-MS fingerprinting was obtained

for all cashew nut oils, and the triacylglycerol information was extracted from the mass spectra

and is shown in Table 6.

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For all samples, the most abundant ions were found for different TAGs, which were mostly

detected as sodium and potassium adduct ions in the form of [TAG + Na] + and [TAG + K]+,

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respectively. These ions were determined in a m/z range of 800 to 950 of the mass spectrum,
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related to TAGs composed of the combination of oleic, linoleic and stearic acids, such as: LA-

LA-LA/O-O-LA, O-LA-LA, O-O-LA, O-O-O/S-LA-O and S-O-O that were detected with m/z
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of 901, 903, 905, 907 and 909, respectively. The most abundant TAG ions are in an agreement
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with the majority FA identified by GC-FID. A screening of oxidation can be seen by the

oxidized TAG ions in form of hydroperoxide, which was detected in a m/z range of 940 to 980,

with the most abundant intensities observed for the oil extracted by SE (F). This behavior
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indicates that the conventional method favors increased TAG oxidation; in the CPE, these ions

were detected in quite low abundances, indicative of the high quality of the extracted oil, as
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previously described by Zanqui et al. [17].

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For all cashew nut oils, the TAG profile presents a very similar composition to that obtained in

another study for some unusual vegetable oils of Brazil, such as Jatropha curcas and Moringa

oleifera, which presented the most abundant ions with m/z of 907 and 909, respectively, related

to O-O-O/S-LA-O and S-O-O [38]. The cashew nut oils also presented a TAG profile similar

to olive oils [39], in an agreement with the FA composition described herein.

3.5 Phytosterol and tocopherols

Tocopherols and sitosterol were quantified in cashew nut oils, as summarized in Table 7. The

highest tocopherols and sitosterol indexes were found in oils obtained by experiments D

(30°C/6% Et) and E (60°C/6% Et) by CPE method, with about 18-19 mg of tocopherols and

60-70 mg of sitosterol per 100 g of cashew oil extracted with n-propane containing 6% ethanol;

the indexes were higher than those obtained by SE method. These data show that the addition

of co-solvent to n-propane helped in the extraction of compounds with bioactive activity, which

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represents more quality to the obtained oil.

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This is clear, noting that the experiments A (30°C/0% Et) and B (60°C/0% Et) extracted the

smallest amounts of tocopherols and were the experiments in which ethanol was not used as
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cosolvent in the extractions with n-propane, indicating that the use of ethanol can favor the

extraction of these compounds by modifying the polarity of the fluid, better solubilizing the
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compounds of interest. Studies have shown that the amount of γ + δ tocopherols in cashew nut
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oils can vary from about 6 to 12 mg of tocopherols per 100 g of oil when extracted by solvents,

values similar to those determined in this study [40].

Results are shown in bar graphs for better understanding in Figure S2 of the supplementary
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material.
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4. Conclusions
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The propane and ethanol solvents mixture has proven attractive for use in the extraction of

edible oil, resulting in satisfactory yields and quality. Obtaining cashew nut oil with pressurized

n-propane and ethanol cosolvent is effective compared to the traditional method, free of toxic

residues. The increase in temperature and percentage of ethanol in the solvent favors obtaining

the highest quality oil in the pressure condition evaluated (8 MPa). The fatty acid composition
was maintained and the amount of tocopherols and sitosterol recovered in the extracts by CPE

were higher than those by SE, especially in the case of the addition of ethanol to propane. The

mathematical model of Sovová adequately adjusted the extraction curves, which contributes to

the understanding of the extraction phases and mass transfer mechanisms. Thus, the extraction

of cashew nut oil with pressurized n-propane and cosolvent is a potential alternative to obtain

high quality oil, free of toxic residues and suitable for later use in the food, cosmetic and

pharmaceutical industries.

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Funding: This work was supported by CAPES (process number 88887.354426/2019-00) and

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Sao Paulo Research Foundation (FAPESP) (process number 2013/19161-4).

Acknowledgments
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To the Universidade Estadual de Maringá for support the development of this research.
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References

[1] M. Kornsteiner, K.H. Wagner, I. Elmadfa, Tocopherols and total phenolics in 10

different nut types, Food Chem. 98 (2006) 381–387.

doi:10.1016/j.foodchem.2005.07.033.

[2] C. Capurso, M. Massaro, E. Scoditti, G. Vendemiale, A. Capurso, Vascular effects of

the Mediterranean diet Part I: Anti-hypertensive and anti-thrombotic effects, Vascul.

Pharmacol. 63 (2014) 118–126. doi:10.1016/j.vph.2014.10.001.

[3] A. Szabo, M. Mézes, C. Hancz, T. Molnár, D. Varga, R. Romvári, H. Fébel,

of
Incorporation dynamics of dietary vegetable oil fatty acids into the triacylglycerols and

ro
phospholipids of tilapia (Oreochromis niloticus) tissues (fillet, liver, visceral fat and

gonads), Aquac. Nutr. 17 (2011) e132–e147. doi:10.1111/j.1365-2095.2009.00743.x.

[4]
-p
N. Tippayawong, C. Chaichana, A. Promwangkwa, P. Rerkkriangkrai, Gasification of

cashew nut shells for thermal application in local food processing factory, Energy
re
Sustain. Dev. 15 (2011) 69–72. doi:10.1016/j.esd.2010.10.001.
lP

[5] M. Herrero, M. Castro-Puyana, J.A. Mendiola, E. Ibañez, Compressed fluids for the

extraction of bioactive compounds, TrAC - Trends Anal. Chem. 43 (2013) 67–83.

na

doi:10.1016/j.trac.2012.12.008.

[6] C.M. Silva, A.B. Zanqui, A.K. Gohara, A.H.P. Souza, L. Cardozo-Filho, J.V.

Visentainer, L.U.R. Chiavelli, P.R.S. Bittencourt, E.A. Silva, M. Matsushita,

ur

Compressed n-propane extraction of lipids and bioactive compounds from Perilla

Jo

(Perilla frutescens), J. Supercrit. Fluids. 102 (2015) 1–8.

doi:10.1016/j.supflu.2015.03.016.

[7] A.B. Zanqui, D.R. Morais, C.M. Silva, J.M. Santos, S.T.M. Gomes, J.V. Visentainer,

M.N. Eberlin, L. Cardozo-Filho, M. Matsushita, Subcritical extraction of flaxseed oil

with n-propane: Composition and purity, Food Chem. 188 (2015) 452–458.
 doi:10.1016/j.foodchem.2015.05.033.

[8] H. Sovová, Rate of the vegetable oil extraction with supercritical CO2—I. Modelling

of extraction curves, Chem. Eng. Sci. 49 (1994) 409–414. doi:10.1016/0009-

2509(94)87012-8.

[9] M.S. Zabaloy, H.P. Gros, S.B. Bottini, E.A. Brignole, Isothermal vapor-liquid

equilibrium data for the binaries isobutane-ethanol, isobutane-1-propanol, and

propane-ethanol, J. Chem. Eng. Data. 39 (1994) 214–218. doi:10.1021/je00014a005.

[10] F. Soxhlet, Die gewichtsanalytische bestimmung des milchefettes, Polytech. J. 232

of
(1879) 461–465.

ro
[11] C.M. Silva, A.B. Zanqui, E.A. Silva, S.T.M. Gomes, L.C. Filho, M. Matsushita,

Extraction of oil from Elaeis spp. using subcritical propane and cosolvent:
-p
Experimental and modeling, J. Supercrit. Fluids. 133 (2018) 401–410.

doi:10.1016/j.supflu.2017.11.006.
re
[12] J.A. Nelder, R. Mead, A simplex method for function minimization, Comput. J. 7
lP

(1965) 308–313. doi:10.1093/comjnl/7.4.308.

[13] L. Hartman, R.C. Lago, Rapid preparation of fatty acid methyl esters from lipids, Lab.

Pract. 22 (1973) 475–476.

na

[14] S.C. Sargi, B.C. Silva, H.M.C. Santos, P.F. Montanher, J.S. Boeing, O.O. Santos

Júnior, N.E. Souza, J.V. Visentainer, Antioxidant capacity and chemical composition
ur

in seeds rich in omega-3: chia, flax, and perilla, Food Sci. Technol. 33 (2013) 541–
Jo

548. doi:10.1590/S0101-20612013005000057.

[15] J.D. Joseph, R.G. Ackman, Capillary column gas chromatographic method for analysis

of encapsulated fish oils and fish oil ethyl esters: collaborative study, J. AOAC Int. 75

(1992) 488–506.

[16] R.F. Zara, E.G. Bonafé, C.A. Martin, N.E. Souza, E.C. Muniz, J.V. Visentainer,
 Preparation of fame by microwave irradiation using boron trifluoride as a catalyst, Am.

J. Anal. Chem. 3 (2012) 288–294. doi:10.4236/ajac.2012.34039.

[17] A.B. Zanqui, C.M. Silva, D.R. Morais, J.M. Santos, S.A.O. Ribeiro, M.N. Eberlin, L.

Cardozo-Filho, J.V. Visentainer, S.T.M. Gomes, M. Matsushita, Sacha inchi

(Plukenetia volubilis L.) oil composition varies with changes in temperature and

pressure in subcritical extraction with n-propane, Ind. Crops Prod. 87 (2016) 64–70.

doi:10.1016/j.indcrop.2016.04.029.

[18] E.C. Cabral, G.F. Cruz, R.C. Simas, G.B. Sanvido, L. V. Gonçalves, R.V.P. Leal,

of
R.C.F. Silva, J.C.T. Silva, L.E.S. Barata, V.S. Cunha, L.F. França, R.J. Daroda, G.F.

ro
Sá, M.N. Eberlin, Typification and quality control of the Andiroba (Carapa guianensis)

oil via mass spectrometry fingerprinting, Anal. Methods. 5 (2013) 1385–1391.

doi:10.1039/c3ay25743f.
-p
[19] R. Haddad, R. Sparrapan, M.N. Eberlin, Desorption sonic spray ionization for (high)
re
voltage-free ambient mass spectrometry, Rapid Commun. Mass Spectrom. 20 (2006)
lP

2901–2905. doi:10.1002/rcm.

[20] M. Du, D.U. Ahn, Simultaneous analysis of tocopherols, cholesterol, and phytosterols

using gas chromatography, J. Food Sci. 67 (2002) 1696–1700. doi:10.1111/j.1365-

na

2621.2002.tb08708.x.

[21] T.H.J. Beveridge, T.S.C. Li, J.C.G. Drover, Phytosterol content in American ginseng
ur

seed oil, J. Agric. Food Chem. 50 (2002) 744–750. doi:10.1021/jf010701v.

Jo

[22] Thermo Electron Corporation; Xcalibur: Quantitative Analysis Software version 2.0.

Waltham, USA, (2006).

[23] NIST, Propriedades termofísicas de sistemas fluidos, (2015).

http://webbook.nist.gov/chemistry/fluid (accessed January 20, 2019).

[24] T.S.C. Li, T.H.J. Beveridge, J.C.G. Drover, Phytosterol content of sea buckthorn
 (Hippophae rhamnoides L.) seed oil: Extraction and identification, Food Chem. 101

(2007) 1633–1639. doi:10.1016/j.foodchem.2006.04.033.

[25] StatSoft, Inc.; Statistica: Data Analysis Software System version 8.0. Tulsa, USA,

(2008).

[26] Stat-Ease, Inc; Design Expert Software version 7.1.3. Minneapolis, USA, (2008).

[27] C.P. Lameira, G.L. V. Coelho, C.G. Mothé, Extração de lipídeos da amêndoa de

castanha de caju com CO2 supercrítico, Ciência e Tecnol. Aliment. 17 (1997) 405–

407. doi:10.1590/S0101-20611997000400012.

of
[28] A.B. Zanqui, C.M. Silva, J.B. Ressutte, D.R. Morais, J.M. Santos, M.N. Eberlin, L.

ro
Cardozo-Filho, J. V. Visentainer, S.T.M. Gomes, M. Matsushita, Brazil Nut Oil

Extraction Using Subcritical n-Propane: Advantages and Chemical Composition, J.

-p
Braz. Chem. Soc. 00 (2019) 1–10. doi:http://dx.doi.org/10.21577/0103-

5053.20190225.
re
[29] A.A.S. Brum, L.F. Arruda, M.A.B. Regitano-d’Arce, Métodos de extração e qualidade
lP

da fração lipídica de matérias-primas de origem vegetal e animal, Quim. Nova. 32

(2009) 849–854. doi:dx.doi.org/10.1590/S0100-40422009000400005.

[30] C.M. Silva, A.B. Zanqui, A.H.P. Souza, A.K. Gohara, M.A. Chaves, S.T.M. Gomes,
na

L. Cardozo-Filho, N.E. Souza, M. Matsushita, Chemometric study of perilla fatty acids

from subcritical n-propane extracted oil, J. Braz. Chem. Soc. 26 (2015) 14–21.
ur

doi:10.5935/0103-5053.20140207.
Jo

[31] A. Quintero-Flórez, L. Sinausia Nieva, A. Sánchez-Ortíz, G. Beltrán, J.S. Perona, The

fatty acid composition of virgin olive oil from different cultivars is determinant for

foam cell formation by macrophages, J. Agric. Food Chem. 63 (2015) 6731–6738.

doi:10.1021/acs.jafc.5b01626.

[32] P.B.F. Biondo, V.J. Santos, P.F. Montanher, O.O.S. Junior, M. Matsushita, V.C.
 Almeida, J. V. Visentainer, A new method for lipid extraction using low-toxicity

solvents developed for canola (Brassica napus L.) and soybean (Glycine max L.

Merrill) seeds, Anal. Methods. 7 (2015) 9773–9778. doi:10.1039/c5ay02243f.

[33] S.A.O. Ribeiro, A.E. Nicacio, A.B. Zanqui, P.B.F. Biondo, B.A. Abreu-Filho, J.V.

Visentainer, S.T.M. Gomes, M. Matsushita, Improvements in the quality of sesame oil

obtained by a green extraction method using enzymes, LWT - Food Sci. Technol. 65

(2016) 464–470. doi:10.1016/j.lwt.2015.08.053.

[34] S.A.O. Ribeiro, A.E. Nicacio, A.B. Zanqui, P.B.F. Biondo, B.A. Abreu-Filho, J. V.

of
Visentainer, S.T.M. Gomes, M. Matsushita, Application of enzymes in sunflower oil

ro
extraction: Antioxidant capacity and lipophilic bioactive composition, J. Braz. Chem.

Soc. 27 (2016) 834–840. doi:10.5935/0103-5053.20150335.

-p
[35] J. Yang, Brazil nuts and associated health benefits: A review, LWT - Food Sci.

Technol. 42 (2009) 1573–1580. doi:10.1016/j.lwt.2009.05.019.

re
[36] A.B. Zanqui, D.R. Morais, C.M. Silva, J.M. Santos, L.U.R. Chiavelli, P.R.S.
lP

Bittencourt, M.N. Eberlin, J. V. Visentainer, L. Cardozo-Filho, M. Matsushita,

Subcritical extraction of Salvia Hispanica L. oil with n-propane: composition, purity

and oxidation stability as compared to the oils obtained by conventional solvent

na

extraction methods, J. Braz. Chem. Soc. 26 (2015) 282–289.

doi:http://dx.doi.org/10.5935/0103-5053.20140278.
ur

[37] G.A. Bataglion, F.M.A. Da Silva, J.M. Santos, M.T. Barcia, H.T. Godoy, M.N.
Jo

Eberlina, H.H.F. Koolen, Integrative approach using GC-MS and easy ambient sonic-

spray ionization mass spectrometry (EASI-MS) for comprehensive lipid

characterization of buriti (Mauritia flexuosa) oil, J. Braz. Chem. Soc. 26 (2015) 171–

177. doi:10.5935/0103-5053.20140234.

[38] F.N. Santos, J.M. Santos, P.R.R. Mesquita, K.B. Oliveira, W.A. Rodrigues, Frederico
 M., Lopes, M.N. Eberlin, Immediate differentiation of unusual seed oils by easy

ambient sonic-spray ionization mass spectrometry and chemometric analysis, Anal.

Methods. 8 (2016) 3681–3690. http://dx.doi.org/10.1039/C5AY03061G.

[39] A.M.A.P. Fernandes, G.D. Fernandes, R.C. Simas, D. Barrera-Arellano, M.N. Eberlin,

R.M. Alberici, Quantitation of triacylglycerols in vegetable oils and fats by easy

ambient sonic-spray ionization mass spectrometry, Anal. Methods. 5 (2013) 6969–

6975. doi:10.1039/c3ay41099d.

[40] Y. Gong, R.B. Pegg, Tree nut oils: properties and processing for use in food, in: G.

of
Talbot (Ed.), Spec. Oils Fats Food Nutr. Prop. Process. Appl., Woodhead Publishing,

ro
Waltham, 2015: pp. 65–80. doi:10.1016/B978-1-78242-376-8.00003-X.

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Figure caption

Fig 1. Response surface that describes the behavior of the extraction yield as a function of

temperature and percentage of ethanol using CPE method.

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Fig 2. Experimental and modeled kinetic curves for the extraction of cashew nut oil by
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compressed n-propane. A: 30°C/0% Et, B: 60°C/0% Et, C: 30°C/6% Et, D: 60°C/6% Et, E:
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45°C/3% Et.
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Fig S1. Bar graphs for major and sum fatty acids. A: 30°C/0% Et, B: 60°C/0% Et, C: 30°C/6%

Et, D: 60°C/6% Et, E: 45°C/3% Et.

Fig S2. Bar graphs for bioactive compounds. A: 30°C/0% Et, B: 60°C/0% Et, C: 30°C/6% Et,

D: 60°C/6% Et, E: 45°C/3% Et.

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Table 1. Factors and levels for the 22 factorial design with central point

Factors Symbol Unit Type Levels

-1 0 +1

Temperature T ºC Numeric 30 45 60

Ethanol Et % Numeric 0 3 6

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 Table 2. Experimental conditions and extraction yield results for the cashew nut oil extraction

using CPE and SE methods.

Temperature Ethanol Solvent Time Yieldb Yield in dry Extraction

Tests Solvent densitya basesc percentaged

(°C) (%) (g cm-3) (min) (%) (%) (%)

A n-Propane 30 0 0.50 60 19.2 21.3 58.0

B n-Propane 60 0 0.46 60 22.3 24.8 67.4

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C n-Propane 30 6 0.53 60 25.2 28.0 76.1

D n-Propane 60 6 0.49 60 27.7 30.8 83.7

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E n-Propane 45 3 0.50 60 23.2±0.1* 25.6 71.6

Ethyl ether-
F
petroleum ether
boiling point - - -p960 33.1±0.6* 36.8 100

*Mean ± standard deviation (n = 3); aValues of density calculated according to NIST, 2015; b100
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times the mass of oil extracted by mass of cashew nut introduced into the extraction column;
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c
Considering 10.05% of moisture in the initial sample; d100 times the percentage of oil

extracted at the end of the total extraction time by the percentage of the oil extracted with ethyl
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ether and petroleum ether.

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Table 3. Data of variance analysis for oil obtained using 22 factorial design for propane

extractions

Terms Sum of squares Degrees of freedom Mean squares F-value p-value

Model 197.42 3 65.81 6580.67 0.0002

T 60.84 1 60.84 6084.00 0.0002

Et 114.49 1 114.49 11449.00 < 0.0001

T.Et 22.09 1 22.09 2209.00 0.0005

Curvature 14.42 1 14.42 1441.71 0.0007

Pure Error 0.020 2 0.01 - -

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Cor Total 211.86 6 - - -

T = temperature; Et: percentage of ethanol

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Table 4. Adjustable parameters for the Sovová model

S tCER tFER KFa KSa

Tests Z W r R2
(g oil/g solvent) (min) (min) (min) (min)

A 1.50 0.01 0.87 0.19 58.19 145.59 5.61 x 10-2 2.51 x 10-4 0.960

B 2.68 0.02 0.87 0.24 28.04 103.81 1.00 x 10-1 6.98 x 10-4 0.970

C 1.10 0.02 0.87 0.24 59.78 126.47 4.14 x 10-2 1.05 x 10-3 0.950

D 2.66 0.03 0.87 0.24 27.12 100.31 9.97 x 10-2 1.35 x 10-3 0.970

E 1.58 0.02 0.87 0.19 55.98 145.27 5.91 x 10-2 7.92 x 10-4 0.984

Z and W: dimensionless parameter of Sovová model, S: solubility, r: easily accessible oil mass,

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tCER: time at which the extraction of the oil from the inside of particles starts, tFER: time at which

the extraction of easily accessible solute ends, KFa: solvent-phase mass transfer coefficient, KSa:

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solid-phase mass transfer coefficient, R2: coefficient of determination.
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Table 5: Quantification and sum of fatty acids for cashew oil

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Experimental conditions 30°C/0% Et 60°C/0% Et 30°C/6% Et 60°C/6% Et 45°C/3% Et SE

Test A B C D E F

pr
RT (min) FA (mg g-1)

11.2 16:0 88.53a±0.29 86.50a±0.53 87.63a±1.97 88.32a±0.97 87.97a±1.87 89.12a±1.28

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11.8 16:1n-7 2.98a±0.08 2.78ab±0.01 2.64b±0.20 2.88ab±0.03 2.69b±0.07 3.00a±0.12

12.8 17:0 0.88b±0.01 1.03a±0.02 1.03a±0.02 1.03a±0.01 1.04a±0.03 1.04a±0.02

Pr
14.6 18:0 90.76b±0.72 94.59a±0.22 96.47a±1.71 93.33ab±0.99 95.94a±1.90 93.82ab±0.75

15.3 18:1n-9 636.59a±4.80 638.70a±1.21 638.11a±1.42 636.03a±8.80 634.66a±1.81 634.04a±1.93

15.5 18:1n-7 3.55b±0.09 3.39b±0.09 3.51b±0.02 3.40b±0.03 3.58b±0.19 4.28a±0.07

16.8 18:2n-6
l 116.19a±0.79 112.58bc±0.15 110.92c±1.88 113.05bc±2.22 112.05bc±0.76 114.58ab±0.63
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17.7 18:3n-6 0.11b±0.02 0.20a±0.02 0.17a±0.01 0.15ab±0.01 0.18a±0.02 0.19a±0.03

18.4 18:3n-3 1.10a±0.09 1.17a±0.01 1.11a±0.02 1.14a±0.05 1.12a±0.01 1.17a±0.04

19.1 20:0 5.54c±0.17 6.36a±0.19 6.32a±0.15 5.99ab±0.03 6.25a±0.18 5.78bc±0.09

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19.8 20:1n-9 1.44b±0.02 1.69a±0.05 1.68a±0.07 1.59a±0.07 1.68a±0.02 1.60a±0.06

22.7 22:0 0.73b±0.02 1.05a±0.06 1.01a±0.05 0.94a±0.03 1.01a±0.03 1.05a±0.07

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25.5 24:0 0.86b±0.09 1.35a±0.11 1.27a±0.03 1.21a±0.04 1.27a±0.08 1.21a±0.20

Sum of FA (mg.g-1)

SFA 187.28b±0.80 190.89ab±0.61 193.73a±2.61 190.82ab±1.39 193.49a±2.68 192.02a±1.50

MUFA 644.56a±4.80 646.56a±1.21 645.94a±1.44 643.90a±8.80 642.62a±1.82 642.92a±1.93

 PUFA 117.39a±0.79 113.94bc±0.15 112.19c±1.88 114.34abc±2.22 113.35bc±0.76 115.94ab±0.63

f
Mean ± standard deviation (n = 3); different letters in the same line indicate a significant difference (p <0.05) by Tukey test; RT: retention time; FA: fatty acids; Et: ethanol;

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SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

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Table 6: Assignment of the main ions detected by EASI(+)-MS for cashew nut oil

[TAG+ [TAG+ [Hydroperoxide+ [Hydroperoxide+

TAG Composition
Na] + (m/z) K] + (m/z) Na] + (m/z) K] + (m/z)

P-LA-LA 877 893 941 957

O-P-O 881 897 - 961

P-O-LA 879 895 - -

LA-LA-LA/O-O-LA 901 917 933 949

O-LA-LA 903 919 - -

O-O-LA 905 921 937 -

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O-O-O/S-LA-O 907 923 971 -

S-O-O 909 925 - -

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P = Palmitic acid; PA = Palmitoleic acid; S = Stearic acid; O = Oleic acid; LA = Linoleic acid;

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Table 7: Bioactive compounds in cashew nut oil

Bioactive compounds
Temperature Ethanol
(mg.100 g-1)
Test Solvent
(γ+δ)
(°C) (%) Sitosterol
Tocopherols

A n-Propane 30 0 10.70c±0.93 37.32d±1.01

B n-Propane 60 0 12.75bc±0.50 42.54cd±3.30

C n-Propane 30 6 13.98abc±4.18 50.62bc±7.67

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D n-Propane 60 6 19.40a±2.52 61.73ab±3.97

18.53ab±3.23 71.92a±4.90

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E n-Propane 45 3

Ethyl ether-

F petroleum

ether
boiling point - -p N/D 57.07b±3.82
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Mean ± standard deviation (n = 3); different letters in the same column indicate a significant difference (p

<0.05) by Tukey test; N/D: Not detected.

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