Enhanced extraction of spent coffee grounds oil using high-pressure CO2 plus ethanol solvents

Industrial Crops & Products 141 (2019) 111723

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Enhanced extraction of spent coffee grounds oil using high-pressure CO2 plus ethanol solvents

T

Micheli Nolasco Araújo, Ana Queren Paladonai Leandro Azevedo, Fabiane Hamerski, ⁎ Fernando Augusto Pedersen Voll, Marcos Lúcio Corazza Department of Chemical Engineering, Federal University of Paraná, CEP 81531-990, Curitiba, PR, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Supercritical carbon dioxide CO2-expanded ethanol Pressurized ethanol Spent coffee grounds Biorefinery

This work reports the extraction of spent coffee grounds (SCG) oil using supercritical CO2 plus ethanol (scCO2+EtOH) solvent in semi-batch process as an alternative approach to the use of organic solvent extraction. In addition, the results are compared to supercritical carbon dioxide (scCO2) and pressurized ethanol (EtOH). Soxhlet was performed and used as a standard extraction method. The extraction yield, fatty acid profile, total phenolic content (TPC), antioxidant activity (AA by ABTS and DPPH methods), phenolic compounds and caffeine content were determined, as well the overall extraction curves. The highest extraction yield of 15.9% was obtained using scCO2+EtOH using an ethanol to SCG ratio mass of (2:1), 80 ºC, 20 MPa in 25 min of extraction time. Major fatty acids present in the oil were linoleic (45%) and palmitic (31%). The oil obtained with scCO2+EtOH (2:1) presented high TPC values (294.47 to 392.96 mg GAE/100 g oil), similar to those obtained by pressurized ethanol extraction. High values of AA by ABTS and DPPH methods were also found in the oil from scCO2+EtOH extraction. The main phenolic acids found were dihydroxybenzoic acid and caffeic acid. The highest concentrations of dihydroxybenzoic (17.66 mg/100 g oil) and caffeic acid (9.36 mg/100 g oil) were obtained with scCO2+EtOH (2:1) at 80 ºC and 10 MPa. The highest caffeine content (711.70 mg/100 g oil) was obtained with scCO2+EtOH (0.5:1) at 60 ºC and 15 MPa. The results obtained in this work provided higher extraction yield when compared to scCO2 and similar to compressed ethanol, however in a shorter extraction time and using lesser organic solvent amounts. The results are promising and demonstrate the technical feasibility of SCG oil extraction using scCO2+EtOH to obtain valuable a product from an agricultural and urban waste biomass.

1. Introduction Coffee is the most consumed worldwide beverage after water and the second largest commodity in stock exchange after petroleum (Akgün et al., 2014; Andrade et al., 2012; Mata et al., 2018). According to International Coffee Organization (ICO) approximately 9.4 million of tons were produced globally in 2018 (ICO, 2018). During the coffee production and processing huge quantities of waste, such as skins and spent coffee grounds (SCG) are generated. Around 650 kg of SCG are generated by 1000 kg of green coffee beans processed (Karmee, 2018). SCG contain many organic compounds as fatty acids, amino acids, lignin, cellulose, hemicellulose, and other polysaccharides, which can be processed and add value to this waste (Campos-Vega et al., 2015). Besides that, it also contains bioactive compounds, particularly alkaloids and polyphenols, and caffeine (major alkaloid), tannins, flavanols, flavones and phenolics acids. Thus, recovering these compounds might

⁎

be interesting for food, pharmaceutical and cosmetic industry applications. However currently, the SCG are being incinerated, generating greenhouse gases and/or allocated into landfills. Thus, SCG have been investigated as a source of value-products to reduce its environmental impact (Mata et al., 2018; Panusa et al., 2013). Considering the quantity and diversity of products that can be obtained from SCG, some authors have studied the feasibility of implementing a biorefinery with SCG as feedstock. According to Karmee (2018) and Mata et al. (2017), a biorefinery based on spent coffee grounds can be a feasible strategy by using a combination of compatible chemical, biotechnological, and thermochemical methods, where biodiesel production (Abdullah and Bulent Koc, 2013; Al-Hamamre et al., 2012; Couto et al., 2009; Döhlert et al., 2015; Kwon et al., 2013; Rocha et al., 2014), extractions of antioxidants to the pharmaceutical and food industry (Ahangari and Sargolzaei, 2013; Andrade et al., 2012; Benavides et al., 2016; Mussatto et al., 2011; Page et al., 2017; Shang

Corresponding author at: Department of Chemical Engineering, Federal University of Paraná, PO Box 19011 Polytechnic Center, Curitiba 81531-980, PR, Brazil. E-mail address: [email protected] (M.L. Corazza).

https://doi.org/10.1016/j.indcrop.2019.111723 Received 5 June 2019; Received in revised form 21 August 2019; Accepted 23 August 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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Pressurized liquid extraction (PLE) is an attractive extraction technique that can promote high efficient extraction in short extraction time using low relatively low amounts of organic liquid solvents (Oliveira et al., 2018). Currently, PLE has been applied to green coffee (Oliveira et al., 2018), wild almond (Balvardi et al., 2015), soybean (Rodrigues et al., 2017) and spent coffee grounds (Shang et al., 2016). The main advantages of PLE is related to the use of high temperatures that promotes high solubility of solutes in the solvent by increasing the diffusional rate of the solute to the solvent and breaking the strong interaction solute-matrix interactions, such as van der Waals bonds, and solute molecules and active sites in the matrix, such hydrogen and dipole-dipole interactions (Oliveira et al., 2018). A gas-expanded liquid (GXL) technique is a mixture of a compressible gas dissolved in a liquid solvent. The CO2-expanded liquids (CXL) are the mostly common class of GXL used, due to the operational advantages of the CO2 already cited (Jessop and Subramaniam, 2007). The CXL is a solvent with gas and liquid characteristics, where CO2 improves the gas solubility and mass transfer, while liquid organic solvents increase the solubility of liquid and solid solutes (Herrero et al., 2017; Siougkrou et al., 2014). In general, CXL can be considered a switchable solvent technique found midway between pressurized liquids and compressed CO2 by increasing the amount of CO2 (Herrero et al., 2017). The CXL has been successfully applied to the extraction of Moringa oleifera (Rodrígues et al., 2016), where the authors reached a extraction yield two-fold higher using 50% of ethanol in the CXL (ethanol and CO2) than with pure scCO2. The main advantages of CXL compared to scCO2 is the possibility of using lower pressure operations, and when compared to PLE the use of lesser solvent amounts (Rodrígues et al., 2016; Toda et al., 2016). However, to the best of our knowledge, a semi-batch technique using CO2-expanded solvent to recovery the oil from SCG has not been reported in the literature. Therefore, this study aimed to evaluate the influence of scCO2+EtOH (CXL) extraction parameters and compare the semi-batch CXL methodology with other green solvents techniques, such as pure scCO2 and pressurized ethanol, in terms of extraction yields, overall extraction curves (kinetics of extraction) and chemical profiles of the extracted oil from spent coffee grounds. Fatty acid (FA) profile, antioxidant activity (AA), total phenolic content (TPC), phenolics compounds and caffeine were also evaluated in order to assess the oil qualify and its potential commercial application.

et al., 2016; Xu et al., 2015), bioethanol (Kwon et al., 2013; Rocha et al., 2014), pellets (Zuorro and Lavecchia, 2012) and tannins (Low et al., 2015), are some examples of by-products that can be obtained from SCG. The crude oil is the most economically valuable and easily recovered compound from SCG (Campos-Vega et al., 2015). For example, there is an increasing interest to produce biodiesel from the residual oil obtained with spent coffee grounds as a feedstock, since this is a sustainable practice for waste reduction and a low-cost source of fatty acids (Al-Hamamre et al., 2012). SCG have potential to biodiesel production since its oil content varies from 12 to 18.3% (Andrade et al., 2012; Couto et al., 2009) and some authors report 85.5% (Al-Hamamre et al., 2012) and 98.61% (Liu et al., 2017) of converted coffee oil to biodiesel by alkali-catalyzed esterification and direct acid transesterification (in situ), respectively. Assuming an oil content of 15% and 92% of converted coffee oil to biodiesel, the production could be approximately 0.85 million tons of biodiesel by year. Furthermore, SCG oil quality can be enhanced to applications in cosmetic and pharmaceutical industry or use it as source of other valuable compounds as caffeine, sterols, terpenes and tocopherols (Campos-Vega et al., 2015). Caffeine is the most studied compound from coffee due its psychoactive effects and the promotion of energetic metabolism (Panusa et al., 2013). Moreover, phenolics compounds from coffee oil have been studied due their beneficial effects to human health, as its protection against chronic degenerative disease (cataracts, macular degeneration, neurodegenerative diseases, and diabetes mellitus) (Ballesteros et al., 2017) and its other bioactivities properties like antioxidant, anti-bacterial, antiviral, anti-inflammatory and anti-carcinogenic activities (Barbosa et al., 2014). Oil extraction is usually performed using organic solvents, however the environment safety rules and the increased public health risk are pushing the industry to seek for alternative extraction methods (Ahangari and Sargolzaei, 2013). Supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), and gas-expanded liquid extraction (GXL) have been considered as green technology and efficient methods to extract compounds from natural matrices due some advantages, such as, elimination of large amount of organic solvents, solvent selectivity by changing the operational conditions to enhanced the recovery a class of specific compounds (Jessop and Subramaniam, 2007; Reverchon and De Marco, 2006; Shang et al., 2016). Extraction of compounds from natural matter is the most studied application of supercritical fluids (SCF). SFE has immediately advantages over traditional extraction methods, such as processing flexibility and elimination of solvent from the extracts in a post-processing stage (Reverchon and De Marco, 2006). SFE is generally performed with carbon dioxide (CO2) due to its low critical pressure (74 bar) and temperature (32 °C), relative non-toxicity, non-flammability, availability in high purity at relativity low cost and easy removal from the extracts (Pourmortazavi and Hajimirsadeghi, 2007). In SFE, the addition of a liquid solvent can improve the extraction efficiency by increasing the solubility of the solute (Pereira and Meireles, 2010). Couto et al. (2009) performed SFE of lipids using supercritical carbon dioxide (scCO2) at different pressures (15.0, 20.0, 25.0 and 30.0 MPa) and temperatures (40, 50 e 55 °C) aiming the subsequent biodiesel production. The highest yield was 15.4% obtained at 25.0 MPa and 50 °C after 3 h of extraction. Moreover, those authors (Couto et al., 2009) evaluated the addition of ethanol as co-solvent corresponding to an ethanol to CO2 mass ratio of 6.5:93.5 (w/w) at 50 °C and 20.0 MPa, which resulted in a reduction of 60% on the extraction time and the total amount of solvent required to achieve an oil extraction yield of 12.9% at the same condition using pure scCO2. Similar results were reported by Andrade et al. (2012), who studied the SCG oil extraction with scCO2 and scCO2 + co-solvent, at 40, 50 and 60 °C and 10 to 30 MPa during 2.5 h. The highest yield from SFE extraction was 10.5 ± 0.2%, while the highest among the extractions yield was 15 ± 2%, obtained by solid-liquid extraction with ethanol.

2. Materials and methods 2.1. Samples Spent coffee ground (SCG) samples were obtained in a local coffee shop (Zaraffa Coffee Shop, Curitiba, Brazil) with initial moisture and volatile compounds of 65.19 ± 0.89%. The raw material was dried in air circulation oven at 40 °C for 48 h until constant moisture and volatile compounds content of 4.94 ± 0.29%. The average particle size was estimate with the method presented by Gomide (Gomide, 1983) using Tyler series sieves in a vertical vibratory sieve shaker, where the milled material retained in each sieve was: mesh 14 (8.05 ± 0.45%), mesh 24 (15.86 ± 0.04%), mesh 28 (14.32 ± 0.20%), mesh 35 (14.68 ± 2.19%), mesh 48 (29.81 ± 5.46%) and mesh 80 (16.38 ± 2.52%). These fractions were blended and packed in plastic bags and stored at 4 °C until the use. 2.2. Determination of moisture and volatile compounds content and particle density Moisture and volatile compounds content (M) were determined by automatic infrared moisture analyzer method (Gehaka, model IVT 200), determined in triplicate according to National Renewable Energy Laboratory (NREL) methodology (Sluiter et al., 2008) using Eq. 1, where minitial is the SCG mass before drying (g) and mfinal is the SCG 2

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the confinement time, the dynamic extraction step with a solvent flow rate around 2.0 mL/min was started, and the extract oil samples were collected in test tubes at predetermined times to obtain the kinetic curves. Samples from the extraction with scCO2+EtOH and pressurized ethanol were placed into an air circulation oven (IKA, Nova Ética, model 400-2) at 70 °C until constant weight. After that, the extracts mass was measured and stored in amber vials under refrigeration at approximately -4 °C. In order to obtain the extraction time and the mass of SCG oil extracted in each run, the extraction was considered finished when the amount of oil extracted in the test tube was equivalent to 1% of the total amount of extracted oil measured in the previous test tubes. The overall extraction yields were also calculated using Eq. 2.

mass after drying (g). Real density was measured using a helium pycnometer (Quantachrome Ultrapyc 1200e), at the Analytical CentralInstitute of Chemistry/Unicamp, Campinas, Brazil. The apparent density was calculated as a direct relation between the total SCG sample mass and the extractor vessel volume.

M=

minitial − mfinal m initial

× 100

(1)

2.3. Soxhlet extraction Soxhlet extractions were used as a standard method to provide a comparation with the extraction with pressurized solvents (CLX, SFE and PLE). These extractions were carried out using 5 g of dried SCG and 150 mL of solvent, where the solvents used were n-hexane (Neon, 99.5% purity), ethyl acetate (Neon, 99.5% purity) and ethanol (Neon, 99.8% purity). Oil extracted was dried in a rotary vacuum evaporator (IKA, Model RV 10 digital). After, it was dried using an air circulation oven (IKA, Nova Ética, model 400-2) until constant mass at 60 °C for nhexane, 65 °C for ethyl acetate (EtAc) and 70 °C for ethanol. In addition, the oil was stored in amber flasks and kept under refrigeration at -4 °C. The extraction yields were calculated using Eq. 2.

Yield ( %) =

Mass of extracted oil (g ) × 100 Mass of spent coffee grounds sample (g )

2.5. Fatty acid profile Fatty acids (FA) profile of the SCG oil samples was performed in a Shimadzu chromatograph (GC 2010 Plus), with a polar capillary column (Shimadzu, model SH-Rtx-Wax, 30 m x 0.32 mm x0.25 μm), flame ionization detector (FID) at 250 °C and split injection mode (1:10) at 240 °C. The oven temperature was programmed to start at 100 °C, and it was maintained for 5 min. Then, it was increased from 100 °C to 240 °C at a rate of 4 °C/min, and maintained at 240 °C for 5 min. The carrier gas was helium at 32.5 cm3/min. The samples were prepared in agreement to official method Ce 2–66 to convert the lipids into fatty acid methyl esters (FAMEs) (AOCS, 1997). FAMEs were identified by comparison with retention times of the standard mixture FAMEs (Supelco, MIX FAME 37, Bellefonte, PA, USA). The quantification of fatty acid was conducted by area normalization procedure. Results were expressed as a percentage of each individual fatty acid present in the sample.

(2)

2.4. Extraction with compressed fluids Extraction with compressed fluids was performed in a laboratory scale unit that has been presented in previous works (Fetzer et al., 2018), in which a few modifications were performed for the PLE extractions. Basically, the experimental setup consisted of a solvent reservoir (CO2 cylinder, for SFE and CXL extractions; or ethanol bottle, for PLE extractions), a thermostatic bath (Nova Ética, Sppencer, model 521-5D) to maintain the temperature of the high pressure syringe pump (ISCO, model 500D, Lincoln, NE 68504, USA) at 10 °C, a jacketed extractor vessel with internal volume of 62.4 cm3 (length 22 cm and diameter of 1.9 cm) coupled to a thermostatic bath (Quimis, model Q214 s), which is used to keep the extractor temperature constant, and a micrometric needle valve o control the solvent mass flow. Different compressed solvents (scCO2, scCO2+EtOH and pressurized EtOH) were used in this study in order to assess the solvent effect in the spent coffee oil recovery from SCG. Suppliers and purity of chemicals used in the extractions at high pressure were: CO2, White Martins S.A., 99.5% purity; and ethanol, Neon, 95% and 99.8% purity. The extractions were carried out at temperatures of 40, 60 and 80 °C and pressures of 10, 15 and 20 MPa. For extractions with scCO2 and pressurized EtOH the extractor was loaded with approximately 17 g of SCG. For scCO2+EtOH (CXL) extractions, the ethanol amount related to the specified ethanol to spent coffee grounds mass ratio (MRES) was mixed with approximately 17 g spent coffee grounds and then the mixture (ethanol and SCG) was loaded into the extractor. The MRES (gethanol:gSCG) studied were 0.25:1 (4.25 g of EtOH and 17 g of SCG), 0.5:1 (8.50 g of EtOH and 17 g of SCG), 1:1 (17 g of EtOH and 17 g of SCG) and 2:1 (34 g of EtOH and 17 g of SCG). To prevent solid particles from entering the piping system, the extremities of the extractor were coated with cotton. All the extractions were performed in two distinct steps defined as static and dynamic extraction stages. The solvent (scCO2, for SFE and CXL extractions, or EtOH, for PLE extractions) was slowly added to the extractor until the desired pressure was reached, and the temperature was previously set to the extraction setpoint. After reaching the desired pressure, the static extraction took place. During this stage, the solid matrix was kept in contact with the solvent phase under the conditions of pressure and temperature of each experiment. The static extraction times (confinement time) evaluated were: 10, 30, 60 and 90 min. After

2.5.1. Iodine value (IV) and saponification value (SV) The iodine value (IV) and the saponification value (SV) of the SCG oil were determined using the fatty acid profile according to AOCS methods Cd 1c-85 and Cd 3a-94. The IV is a measure of the average amount of unsaturated fats and oils, expressed in g of iodine absorbed per 100 g of sample (g I2/100 g oil) (Knothe, 2002). The IV values were calculated with Eq. 3, using the percentages (in mass basis) of the fatty acid found in SCG oil.

IV = ( % hexadecenoic acid × 0.99760) + ( % octadecenoic acid × 0.8986) + ( % octadienoic acid × 1.810) + ( % octadecatrienoic acid × 2.735) + ( % eicosenoic acid × 0.8175) + ( % docosenoic acid × 0.7497) (3) The saponification value (SV) is defined as the amount of alkali needed to saponify a defined sample weight. It is expressed in mg of potassium hydroxide per g of sample and specifies the total fatty acid content in oil (mg KOH/g oil). The average molecular weight of fatty acids in a lipid system, as well as the number of ester bonds per gram of sample, can be derived from SV (Al-Hamamre et al., 2012). The SVs were calculated with Eq. 4, where mmw is the mean molecular weight (sum fractional), 56.1 is the molecular weight of KOH and 92.09 molecular weight of glycerol.

SV =

3 × 56.1 × 1000 [(mmw × 3) + 92.09] − (3 × 18)

(4)

2.6. Total phenolic content (TPC) Total phenolic content (TPC) in the SCG oil samples obtained by the different extraction methods was determined according to the Folin–Ciocalteu method (Singleton and Rossi, 1965). Briefly, the oil samples were weighed, approximately 150 mg of each sample, and then 1.5 mL of methanol chromatographic grade were added. This mixture was vigorously shacked for 5 min and centrifuged for 10 min at 3

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an equipment Agilent 1200 Series, with a diode array detector (DAD), quaternary pump, automatic injector, degassing system and a column (Zorbax Eclipse XDB-C18 (4.6 mm diameter, 150 mm in length and 5 μm particles). The elution of the compounds was performed in gradient mode between the mobile phase A and B, with constant flow rate of 0.9 mL/min. Phase A was composed of the volumetric ratio of 95% purified water in a Milli-Q system, 2% formic acid PA (Vetec, 85% purity) and 3% acetonitrile (Panreac, 99% purity). Phase B was composed of 48% water, 2% formic acid and 50% acetonitrile. Both phases were previously filtered in a vacuum system, with a Nylon membrane filter with a diameter of 47 mm and a porosity of 0.45 μm. Subsequently, the gases dissolved in the mobile phase were eliminated in an ultrasonic bath, 154 W of power (Unique, model USC-1880 A) at 20 °C for 10 min. The gradient composition of the chromatographic run was initially 90% for phase A and 10% for phase B, ranging to 10% of A in 25 min. At 27 min the composition of the mobile phase returned to the initial condition of 90% A and 10% B. The wavelength used for all compounds analyzed was 280 nm. The compounds identification was performed based on comparison of retention times and absorption spectra between standard peaks (dihydroxybenzoic acid, caffeic acid, pcoumaric acid, ferulic acid and caffeine obtained from Sigma-Aldrich, St. Louis, MO, USA) and the peaks obtained on the chromatograms of the samples. The quantification was performed by external calibration. Results were expressed as milligrams of each individual compound per 100 g of oil (mg/100 g oil).

3000 rpm. The methanol phase was removed and used to determine the TPC. In this procedure, aliquots of 0.05 to 0.2 mL were prepared as described above, and methanol was added to complete the volume up to 0.5 mL and mixed with 2.5 mL of Folin–Ciocalteu phenol reagent (diluted with distilled water to 0.2 mol/L). After 4 min, 2.0 mL of solution of sodium carbonate (75 g/L) was added. The mixture was kept at ambient temperature and protected from light for 120 min. Subsequently, the absorbance was measured in a spectrophotometer at 760 nm. The quantitative results were calculated using an analytical curve of gallic acid and are expressed as mg of gallic acid equivalent per 100 g of oil (mg AGE/100 g oil). 2.7. Antioxidant activity (AA) 2.7.1. ABTS method Antioxidant activity (AA) was determined from 2,2-azino-bis-(3ethylbenzotiazoline-6-sulfonic acid) (ABTS) with radical scavenging. It was performed based on the procedure described by Re et al. (Re et al., 1999). ABTS was dissolved in water to a 7 mmol/L final concentration. This solution (5 mL) was mixed with 88 μL potassium persulfate solution (140 mmol/L) and then incubated in the dark for 16 h at room temperature to produce a stock solution of the radical cation (ABTS•+). The ABTS•+ working solution was prepared by diluting the stock solution with absolute ethanol until reaching an absorbance of 0.700 ± 0.020 at 734 nm. For the sample analyses, aliquots of methanolic solutions prepared as previously described (procedure of TPC), and methanol up to 100 μL was added to amber bottles and mixed with 3.9 mL of the ABTS•+ radical cation working solution (A734 nm = 0.700 ± 0.020). The resulting solution was kept in a dark environment for 6 min. After that time the absorbance was measured at 734 nm with a UV–vis spectrophotometer (model UV-1100, Pro-Analysis). Trolox (Sigma-Aldrich Co, St. Louis, USA) was used as an antioxidant standard for construction of the standard curve. The results of AA were expressed as μmol of Trolox equivalent antioxidant capacity (TEAC) per 100 g of oil (μmol TEAC/100 g oil).

2.9. Statistical analysis In this work the results obtained were analyzed using the software Statistica 7.0® (Analytical Software, Tallahassee, FL, USA), and each response was evaluated independently. Experimental data were analyzed for variance (ANOVA), and the average comparison considering the expanded uncertainties with 95% of confidence level calculated from triplicate results and assumed the same value for all other conditions in the same group of experiments (scCO2, PLE and scCO2 + EtOH groups of experiments).

2.7.2. DPPH method Antioxidant activity determination by the DPPH (2,2-diphenyl-1picrylhydrazyl) method was performed according to the method proposed by Kalantzakis et al. (2006) with minor modifications. Samples of SCG oil were prepared as described in the determination of TPC. Subsequently, volumes of 0.05 to 0.1 mL of this methanol solution were added to 3.9 mL of freshly prepared solution of DPPH in methanol (0.06 mmol/L) and stirred until homogenization. The resulting solution was kept in a dark environment for 60 min. After that time the absorbance was measured at 515 nm on a UV–vis spectrophotometer (model UV-1100, Pro-Analysis). The quantification was performed using a Trolox analytical curve, and the results were expressed as μmol of Trolox equivalent antioxidant capacity (TEAC) per 100 g of oil (μmol TEAC/100 g oil).

3. Results and discussion 3.1. Raw material and Soxhlet extraction SCG samples used in this work presented an average particle diameter, real density and apparent density of 0.466 ± 0.003 mm, 1.23 g/ mL and 0.27 g/mL, respectively. The results of the Soxhlet extractions are presented in Table 1. The overall extraction yield obtained with nhexane was around 15%, value in agreement with the literature that reports values within 12 to 18.3% (Ahangari and Sargolzaei, 2013; Akgün et al., 2014; Andrade et al., 2012; Barbosa et al., 2014; Couto et al., 2009; Melo et al., 2014). Such variation in the extraction yield might be attributed to the difference in the coffee cultivated depending of the local, climatic conditions, harvest time and drying method related to each region (Jenkins et al., 2014). Furthermore, the composition of the Coffea arabica and Coffea robusta (most commercialized species) are significantly different, whence total lipids in blends formed from these species must vary even more.

2.8. Phenolic compounds and caffeine Phenolic compounds and the caffeine of the SCG oil samples were determined by high performance liquid chromatography (HPLC), using Table 1 Results for Soxhlet extraction of SCG oil with different solvents. Run

Solvent

BTa (°C)

Polarity indexb

This work# Couto et al. (2009) Extraction yield (%)

Andrade et al. (2012)

Ahangari and Sargolzaei (2013)

S1 S2 S3

n-Hexane Ethyl acetate Ethanol

69 71 79

0.0 4.4 5.2

14.52 ± 0.52 15.04 ± 0.60 15.64 ± 0.17

12 ± 1 11.8 15

16.7 ± 0.1 – –

a BT: boiling temperature obtained from Efthymioupulos et al. (2018); calculated from triplicate results.

b

18.3 – –

Andrade et al. (2012); #Average and expanded uncertainties with 95% of confidence level

4

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temperature and pressure variation, which explain the univariant extraction yield.

Results presented in Table 1 indicate a tendency to increase the yield by increasing the solvent boiling temperature and the polarity index, as expected. The highest yield was 15.64 ± 0.52% obtained with ethanol, which has the highest boiling temperature and higher polarity among the solvents studied in this work. The high yields at high temperatures are due to the breakdown of the cellular structure of the coffee, which makes the solutes more available, thus increasing the solubility of these compounds in the solvent (Efthymiopoulos et al., 2018). Besides provided the highest extraction among the organic solvents evaluated in Soxhlet extraction, the ethanol also presents lower risks to human health and the environment when compared to n-hexane and other organic solvents (Shang et al., 2016), making it desirable for applications in the food, pharmaceutical and cosmetic industries. Therefore, the ethanol was selected as the liquid solvent for the further extraction presented in this work.

3.2.1. Overall extraction curves using scCO2 Fig. 1 depicts the extraction curves of scCO2 extraction. The influence of the pressure is better evidenced by comparing the results at 40 °C, where the yield increased from 0.82% (obtained at 165 min extraction) to 12.19% at 20 MPa (295 min extraction). According to Akgün et al. (2014) the pressure is the most important parameter that influences positively the scCO2 extraction yield, where high yield values can be obtained through increasing pressure that consequently increases the density and solvation power of the CO2 (Ahangari and Sargolzaei, 2013). Influence of the temperature on the extractions with scCO2 is more complex than pressure due to the combination of two variables: density and vapor pressure. The vapor pressure of the solutes increases with the temperature, increasing the solubility in compressed CO2, however, the density of the solvent decreases by increasing the temperature diminishing the solutes solubility. The main effect depends on the magnitude of these variables for each system (Pourmortazavi and Hajimirsadeghi, 2007). In Fig. 1, at pressures from 10 to 20 MPa, it is observed a negative effect on extraction yields when the temperature increased from 40 °C to 80 °C. Couto et al. (2009) also observed a negative effect of the temperature on the extraction at pressures up to 20 MPa, and reported that only at high pressures (25 and 30 MPa) the density of scCO2 decreased moderately with increasing temperature and the effect of the increase in the solute vapor pressure prevailed resulting in increasing in the overall extraction yield. Extraction yield obtained in Soxhlet with n-hexane (14.52 ± 0.52%) was higher than the highest yield (12.19 ± 0.21%) obtained with scCO2 (40 °C and 20 MPa in 4.9 h of extraction). Couto et al. (2009) obtained similar results, reaching an extraction yield with n-hexane in Soxhlet of 18.3% and the maximum yield in SFE was 15.4%, at pressures of 15 and 30 MPa and temperatures of 40 and 60 °C in 3 h of extraction. In addition, the effects of pressure and temperature were the same as those observed in the present study. Andrade et al. (2012) also reported pressure and temperature effects as those presented in this study. Those obtained yields of 0.43–10.5 % of SCG oil using scCO2 at pressures of 10–30 MPa and temperatures of 40–60 °C with 2.5 h of extraction, while the yield obtained with n-hexane in Soxhlet was 12%. Results regarding SCG extraction with scCO2 obtained in this work and those presented in the literature are indicating that high-pressure

3.2. SCG extraction with scCO2 and pressurized ethanol (PLE) In order to compare the scCO2 + EtOH extraction approach used in this work (CXL), which is the main subject, extractions using scCO2 and compressed ethanol (PLE) were also performed and the results of the extraction curves are presented in Figs. 1 and 2. All experiments of scCO2 and PLE were performed using 30 min of static extraction time (confinement period). The overall extraction curves of different extraction technique will be further discussed and compared later, for instance an analysis of main factor of each extraction is presented. To compare the extraction yield obtained in scCO2 and PLE the same extraction time was set for each process, 145 and 35 min for scCO2 and PLE, respectively, and these results are presented in Table 2 along with the experimental conditions evaluated. ScCO2 results were statistically significant (p ≤ 0.05) when considering the CO2 density as the main factor, showing as expected that the extraction yield increases as the solvent density is increased, which was obtained at the lower temperature and higher pressure evaluated. The supercritical fluid-based extraction is affected by the thermodynamic conditions because at the critical region the fluid exhibits a liquid-like density and a much more increased solvent capacity that is pressure-dependent. For PLE, extraction yield reached high values, comparable to the results obtained in Soxhlet with ethanol (Run S3, Table 1), and both temperature and pressure evaluated were statistically non-significant (p ≥ 0.05) at 35 min of extraction, which means that all conditions achieved the same extraction conditions regardless of temperature and pressure variation. It can be seen in Table 2 that the solvent density changed a little with

Fig. 1. Experimental overall extraction curves for spent coffee ground (SCG) oil with scCO2 at different temperature (40 °C, 60 °C and 80 °C) and pressure (10 MPa, 15 MPa and 20 MPa). 5

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Fig. 2. Experimental overall extraction curves for spent coffee grounds oil with pressurized ethanol at different temperature (40 °C, 60 °C and 80 °C) and pressure (10 MPa, 15 MPa and 20 MPa).

ethanol (15.64%). Similar effects of pressure and temperature on the extraction yield of green coffee oil were reported by Oliveira et al. (2018), where temperature (50–74 °C) and static extraction time (3–9 min) were the only variables that had significant effect on PLE. At a fixed pressure of 10.30 MPa, Oliveira et al. (2018) obtained extraction yields of 6.60–9.78 % of green coffee oil, higher than the yield of 7.57% obtained with n-hexane in Soxhlet. Even though the PLE results obtained in this work are very promising for the oil extraction from SCG, it is worth mention that this technique used a large amount of solvent and it separation when required is energy consuming operation, which is normally performed by either distillation or evaporation to get concentrated extracts for further applications. Therefore, a process that demands lower amounts of liquid solvent might be a potential strategy.

conditions are need to recover most of the oil present in this raw material, and indeed the addition of an organic cosolvent or a combined mixture of scCO2 plus organic solvent (as ethanol) can be a feasible option, since lower pressure conditions would be requited. 3.2.2. Overall extraction curves of SCG using PLE The overall extraction curves for PLE extraction using ethanol under the same experimental conditions used in the extraction with scCO2+EtOH (Table 2) and 30 min of static extraction are presented in Fig. 2. For PLE, the highest extraction yields (15.12% and 15.74%) were obtained at the highest temperature of 80 °C and 10 and 20 MPa, respectively. Similar yields for both pressures at the same temperature were expected, since EtOH is a non-compressible fluid and its density is slightly affected by varying the pressure, as discussed on PLE results. In Fig. 2, the temperature increase from 40 °C to 80 °C, at both fixed pressures, increased both the extraction yield and the initial extraction rate. In PLE, high temperatures improve the contact of the solvent with the solute, therefore the extraction is enhanced due to the increase in the extraction rates, the mass transfer and the solubility of the compounds in the solvent, and also due to the decrease of the surface tension and viscosity (Shang et al., 2016). Extraction yields for PLE using ethanol at 80 °C were higher than the overall yields obtained in Soxhlet with n-hexane (14.52%), and in the same order to the yields obtained with ethyl acetate (15.04%) and

3.3. Extraction using scCO2+EtOH The yields obtained for the extractions with scCO2+EtOH at different conditions of pressure, temperature, ethanol to SCG mass ratio (MRES) and confinement time are shown in Table 3. In addition, Table 3 presents additional experiments for scCO2+EtOH extraction using ethanol at 95% of purity (around azeotropic ethanol). The highest overall yield among all conditions performed was 15.87% (Run 19), obtained using scCO2+EtOH with a MRES of 2:1 at 80 °C and 20 MPa in

Table 2 Experimental conditions and results for SCG oil extraction using scCO2 (SFE) and pressurized ethanol (PLE). m0, solvent b (g)

msolvent c (g)

Dynamic extraction time (min)

Extraction yield (%)

0.628 0.222 0.840 0.593 0.604

51.27 23.01 64.40 48.83 50.20

303.98 174.20 578.38 284.70 314.51

145 145 145 145 145

0.69 0.11 6.25 1.67 0.82 ± 0.21$

0.780 0.742 0.787 0.750 0.743

72.77 70.30 78.03 75.49 72.77

87.54 55.94 72.51 88.92 87.54

35 35 35 35 35

13.74 15.12 13.72 15.29 14.25 ± 0.39$

Run

Solvent

T (°C)

P (MPa)

ρ solvent

SFE 1 2 3 4 5

scCO2 scCO2 scCO2 scCO2 scCO2

40 80 40 80 60

10 10 20 20 15

PLE 6 7 8 9 10

EtOH EtOH EtOH EtOH EtOH

40 80 40 80 60

10 10 20 20 15

a

(g/mL)

a CO2 and ethanol densities obtained from the NIST database (NIST, 2018) and (Green and Perry, 2007) calculated at pressure and temperature conditions of the experiments (T and P); b Mass of solvent (CO2 or ethanol) used in the static extraction; c Mass of solvent (CO2 or ethanol) used in the dynamic extraction; $ Average and expanded uncertainties (with 95% of confidence level) calculated for SFE and PLE extraction yields in triplicate experiments.

6

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Table 3 Experimental conditions and results for SCG oil extraction using scCO2+EtOH (CXL). T (°C)

P (MPa)

IPECa (wt%)

m0, EtOH b (g)

m0, CO2 c (g)

msolvent d (g)

CTe (min)

Extraction time (min)

Extraction yield (%)

Experimental design with ethanol 99.8% 11 scCO2+EtOH (0.5:1) 12 scCO2+EtOH (0.5:1) 13 scCO2+EtOH (0.5:1) 14 scCO2+EtOH (0.5:1) 15 scCO2+EtOH (0.5:1)

40 80 40 80 60

10 10 20 20 15

11.89 21.96 10.23 13.25 13.72

6.93 7.31 6.92 7.31 7.53

51.33 25.98 60.72 47.85 47.35

18.,71 138.50 146.95 108.00 28.63

30 30 30 30 30

10 75 75 55 15

10.35 0.46 12.85 5.59 9.13 ± 0.34f

16 17 18 19 20

40 80 40 80 60

10 10 20 20 15

57.22 61.54 55.99 65.78 59.95

33.43 33.43 34.00 32.26 33.76

24.99 20.89 26.72 16.78 22.55

46.04 19.38 88.15 49.03 47.71

30 30 30 30 30

25 10 45 25 25

13.42 12.62 13.82 15.87 14.14 ± 0.16f

Additional experiments with ethanol 99.8% 60 21 scCO2+EtOH (0.25:1) 22 scCO2+EtOH (1:1) 60 23 scCO2+EtOH (0.5:1)* 40

15 15 10

8.28 28.10 14.65

4.41 16.32 16.13

48.84 41.75 93.99

66.77 28.62 41.49

30 30 30

35# 15 22.5

2.62 12.46 13.53

15 15 15 15 15 15 15

27.29 21.95 27.53 26.31 6.11 12.35 60.02

15.63 13.29 15.80 15.85 3.37 6.92 33.63

41.64 47.26 41.60 44.40 51.75 49.13 22.4

38.58 38.78 38.69 28.88 29.55 24.25 38.38

10 30 60 90 30 30 30

20 20 20 15 15 12.5 20

11.76 11.54 ± 0.55f 11.00 11.55 0.99 7.40 13.61

Run

24 25 26 27 28 29 30

CXL (MRES)

scCO2+EtOH scCO2+EtOH scCO2+EtOH scCO2+EtOH scCO2+EtOH

(2:1) (2:1) (2:1) (2:1) (2:1)

Additional experiments with 95% scCO2+EtOH (1:1) scCO2+EtOH (1:1) scCO2+EtOH (1:1) scCO2+EtOH (1:1) scCO2+EtOH (0.25:1) scCO2+EtOH (0.5:1) scCO2+EtOH (2:1)

60 60 60 60 60 60 60

a Initial ethanol mass fraction (wt%) in the scCO2+EtOH mixture in the CXL extraction; b Mass of ethanol used in the static extraction; c Mass of CO2 used in the static extraction; d Mass of solvent (CO2 or ethanol) used in the dynamic extraction; e CT: confinement time; f Average and expanded uncertainties (with 95% of confidence level) values in triplicate experiments; # Extraction did not reach the theoretical yield of extractable solute; * Sequential extraction.

ethanol addition above MRES of 0.5:1 increased on average 90% the extraction yields when compare to extraction with pure scCO2 showing that the ethanol addition contributed to enhance the extraction efficiency. For extractions with MRES of 2:1 (Runs 16–20), where IPEC values ranged from 57.22 to 65.78 wt% ethanol in CXL mixture. The extraction yields at condition with high amount of ethanol the system behaved likely PLE system reaching yields within 12.97 to 15.97%, while pressurized ethanol the extraction yields were from 13.92 to 15.74 %. However, it is worth to mention that smaller amounts of ethanol were used in the extractions with scCO2+EtOH (2:1) (32.26 to 33.7 g of ethanol) than in extractions with pressurized ethanol (150.54 to 164.41 g of ethanol). The scCO2+EtOH (2:1) extraction performance was similar to the extraction with pressurized ethanol, where the highest yields were obtained by increasing the temperature to a fixed pressure, except for the extraction at 80 °C and 10 MPa. In Table 3 can be observed that for extractions using scCO2+EtOH, the initial percentage of ethanol in the semi-batch CXL mixture (IPEC) varied for the same MRES. The addition of liquids into the extractor vessel can be performed in two ways: as a mixed fluid in the pumping system using a second pump and a mixing chamber; or adding the liquid direct into the sample before the extraction (Pourmortazavi and Hajimirsadeghi, 2007). In this work, ethanol was added as a static cosolvent, being mixed with the matrix and rapidly added to the extractor, and after that the CO2 was added until reach the pressure fixed for the extraction. As CO2 density depends on temperature and pressure the CO2 amount inject varied according to the extraction conditions, even though the MRES was constant. Among the advantages of using a system with the static method, stand out the simplicity to operate the extraction equipment and energy saving related to the use of only one pump for solvent flow and pressure control during the dynamic extraction. In order to study the influence of the confinement time (static extraction) on the extraction yields, experiments were performed at 60 °C, 15 MPa, MRES 1:1 and using ethanol 95% purity (5% water, wt%) at 10, 30, 60 and 90 min for the static extraction period (Run 24–27). As

25 min extraction. Compared to the extraction using scCO2 (Run 4) at same condition (80 °C and 20 MPa), this result represents an increase of 14.20 p.p. in the extraction yield and 83% reduction in the extraction time. In addition, the mass of CO2 used during the static extraction decreased from 48.83 to 16.78 g of CO2 and the consumption of solvent during the dynamic step (solvent) was 284.70 to 49.03 g of CO2. Results obtained with scCO2+EtOH (2:1), Run 19 (80 °C and 20 MPa), were better in terms of ethanol consumption compared to the pressurized ethanol extraction from Run 9 (80 °C and 20 MPa). Although the overall extraction yields were similar, the comparison between the extraction with pressurized ethanol and scCO2+EtOH (2:1) shows that the ethanol consumption during the static extraction (m 0, EtOH ) decreased from 75.49 to 32.26 g and during the dynamics extraction stage the CO2 (Run 19) was lower than the mass used in the pressurized ethanol extraction (Run 9). A disadvantage of the extraction with scCO2+EtOH is the use of two solvents, but the CO2 at ambient pressure becomes gas and it is easily separated from the extracts, and the ethanol separation might be unnecessary if the SCG oil will be used for biodiesel production, for example, since ethanol is used in the transesterification reaction (Döhlert et al., 2015). However, if the solvent removal is required, ethanol separation from the extracts obtained with pressurized ethanol will be more expensive than from the extraction with scCO2+EtOH because the ethanol volume is higher in the first case. In general, the results in Table 3 indicates the increase in extraction yields obtained in the semi-batch CXL process with the increase in the ethanol to spent coffee grounds mass ratio (MRES). The highest yield variation related to MRES of increase from 0.5:1 to 2:1 was 12.16 p.p., obtained at condition of 80 °C and 10 MPa. For the extractions with scCO2+EtOH, the effects of pressure and temperature on the yield showed a significant variation with the MRES variation. In extractions using MRES of 0.5:1 (Runs 11–15) the initial percentage of ethanol in scCO2+EtOH mixture (named IPEC) ranged from 10.23 to 21.96 wt% ethanol, indicating a predominant presence of scCO2 in the extraction vessel. As observed for the scCO2 extractions, low temperature and high pressure resulted in higher yields. However, 7

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Fig. 3. Experimental overall extraction curves for spent coffee grounds oil with scCO2+EtOH at 60 °C, 15 MPa and different temperature ethanol to SCG mass ratio (MRES = 0.25:1, 0.5:1, 1:1 and 2:1).

In Fig. 3, increasing the MRES values in CXL extractions resulted in higher overall extraction yields. Extraction with scCO2+EtOH (2:1) showed a yield of 14.14%, a result superior to the yield of 9.13% obtained with scCO2+EtOH (0.5:1). However, the amount of ethanol used in the MRES of 0.5:1 was 4 times lower than that used in the extraction with 2:1 and the extraction time was 40% lower, which implies in lesser solvent during the dynamic extraction. In Fig. 3, increasing MRES from 0.25:1 to 1:1 also increased the initial extraction rate. However, the initial extraction rate at MRES of 2:1 is lower than the initial extraction rate at 1:1. For the MRES conditions of 2:1, higher mass transfer resistance caused by the ethanol excess related to the solids in extraction bed probably affected the CO2 diffusion into the solvent + solids phase leading to lower extraction rates. Similar effects of increasing the percentage of ethanol in the CXL mixture (IPEC) were observed by Rodrígues et al. (2016). Those authors observed that at 50 °C and 7 MPa using 50% ethanol (lower percentage of ethanol studied) the yield of Moringa oleifera extract was 2 times higher than the yield for the SFE extraction with scCO2 at same experimental condition in 200 min of extraction. Analyzing the percentage of ethanol at a fixed extraction

the extraction yields obtained with different confinement times were similar, 30 min was adopted as the confinement time for extractions to provide a longer contact time of solvents and matrix during the static extraction stage. Fig. 3 depicts the overall CXL extraction curves with scCO2+EtOH, at different MRES (0.25:1, 0.5:1, 1:1 and 2:1), using 99.8% purity ethanol, 60 °C and 15 MPa, which can be compared with the overall extraction curves with scCO2 and pressurized ethanol (PLE) at same experimental condition. From Fig. 4 it can be observed that the addition of ethanol increased the extraction yield from 0.98% (55 min extraction) using scCO2 (Run 5) to 2.62% (35 min extraction) using scCO2+EtOH (0.25:1) (Run 21). Thus, the extraction yield using scCO2+EtOH with 8.28% of ethanol in the CXL mixture (IPEC) was 2.6 times higher than the extraction yield with scCO2 at same condition. The highest yield in scCO2+EtOH extraction was 14.14% obtained with a MRES of 2:1, which is around the same extraction yield obtained with pressurized ethanol (14.25%). However, it worth mentioning again that the extraction with CXL has the advantage that lower amounts of ethanol were used when compared to PLE.

Fig. 4. Experimental overall extraction curves for spent coffee grounds oil with scCO2+EtOH at MRES of 0.5:1, different temperature (40 °C, 60 °C and 80 °C) and pressure (10 MPa, 15 MPa and 20 MPa). 8

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Fig. 5. Phase diagram for CO2(1) + ethanol(2) at 40 °C (black lines), 60 °C (red lines) and 80 °C (blue lines). Continuous and dashed lines are the saturated liquid and vapor phases, respectively. Diagram calculated using the Peng-Robinson EoS with the binary interaction parameters (k12 = 7.8332 × 10−2 and l12 = -3.0999 × 10−2) as presented by Araújo et al. (2012).

experimental conditions (0.222 g/mL). Thus, the CXL solvent phase formed by CO2 and ethanol had insufficient solvating power to solubilize the spent coffee ground oil. Therefore, the results for this condition (80 °C and 10 MPa) with scCO2+EtOH and MRES of 0.5:1 reached around the same values of the extraction yield (Run 2) and initial extraction rate obtained with pure scCO2 (Fig. 1). The overall curves for scCO2+EtOH extraction with MRES of 2:1, presented in Fig. 6, show a different behavior when compared to the curves in Fig. 4. For all conditions it is observed that after the initial extraction rate step the oil recovery was abruptly interrupted and the extraction curves presented different behavior at different temperatures (40 and 80 °C). At 40 °C the yields were around the value comparing the results at 10 and 20 MPa, like that observed in the extraction with pressurized ethanol under the same conditions of temperature and pressure. While at 80 °C a decrease is observed by decreasing the pressure. This occurs because during the dynamic extraction at 80 °C and 10 MPa the CXL mixture goes through the VLE region during the extraction path and the solvation power is not enough to achieve the yield obtained at 80 °C and 20 MPa, as observed in the extraction with pressurized ethanol under the same conditions. However, even the phase diagram presented in Fig. 5 is indicating phase separation at 80 °C and 10 MPa, the ethanol excess in the extractor vessel resulted in oil recovery of 86.91%. Regarding the extraction curves using scCO2+EtOH (2:1) presented in Fig. 6, it is observed that lower pressure (10 MPa) resulted in higher initial extraction rates. In addition, the highest extraction rate at 2:1 RMEB were obtained at 80 °C, whereas for the extraction using scCO2+EtOH (0.5:1) the highest initial rates were obtained at 40 °C. This is indicating that in the extraction with scCO2+EtOH (2:1) the effects related to the solubility of compounds in ethanol are more significant, while for the extraction using scCO2+EtOH at 0.5:1 (Fig. 4) the effect of increasing density and solvation power of CO2 by decreasing temperature are more important. Fig. 7 depicts a comparison of overall extraction curves obtained at different process conditions. For extraction using ethanol along with scCO2 the initial extraction rate is similar even at different process condition (temperature and pressure), showing that the extraction is essentially controlled by the ethanol presence and the interactions between the organic solvent and solutes in the raw material. However, overall extraction yields reached different equilibrium values. The

time, Rodrígues et al. (2016) found that increasing the ethanol fraction from 50 to 60% resulted in increased yield. While the increase of 60 to 70% ethanol decreased the extraction yield. Such behavior may be an indicative of the decrease in the initial extraction rate at high ethanol concentrations observed in the present work, which requires a longer extraction time to obtain higher extraction yields. Fig. 4 shows the overall CXL extraction curves using scCO2+EtOH at MRES of 0.5:1 with 30 min of confinement time. At this mass ratio, the extraction behavior was different depending on the experimental condition of temperature and pressure. For extractions of Run 11 (40 °C and 10 MPa), Run 14 (80 °C and 20 MPa) and Run 15 (60 °C and 15 MPa), in Fig. 5, after the initial extraction rate step the oil recovery was suddenly interrupted. This indicates that the first minutes of the extraction are controlled by the interaction between ethanol and the solute molecules, and then the oil recovery is abruptly reduced when the ethanol concentration in the extractor vessel decreases significantly (Fetzer et al., 2018). For the extraction at 40 °C and 20 MPa (Run 12) the three periods of supercritical extraction (CER, FER and DCR) (Pereira and Meireles, 2010) were observed. At this condition, high CO2 density allows the extraction of SCG oil even after the ethanol concentration has decreased. In the extraction at 80 °C and 10 MPa (Run 13), the lowest yield among the experimental conditions was obtained during a single extraction step with a constant and low extraction rate. The different behavior among the extraction curves in Fig. 4 can be explained by the extraction path within the phase diagram of the CO2+ethanol mixture, as shown in Fig. 5. During the dynamic extraction, the pressure is constant and the CO2 concentration in the extractor vessel increases with the extraction time, because the ethanol is also “extracted” from the vessel by the scCO2 carrying the solutes (extracts) along with them. At 10 MPa and 80 °C, as the CO2 concentration inside the extractor increases, the CXL mixture tends to the vapor-liquid (VLE) region. For the other experimental conditions, at the beginning of the extraction the CXL mixture is in a single phase, above the VLE region, and even the scCO2 switches the ethanol the systems remain in the one-phase region. As the extraction time increases, the CO2 molar fraction increases and the CXL mixture tends to the supercritical region. However, the solvent phase is always above the saturation line of the VLE. The lowest yield for the MRES of 0.5:1 was obtained at 80 °C and 10 MPa. In this condition CO2 has the lowest density among the 9

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Fig. 6. Experimental overall extraction curves for spent coffee grounds oil with scCO2+EtOH at MRES of 2:1, different temperature (40 °C, 60 °C and 80 °C) and pressure (10 MPa, 15 MPa and 20 MPa).

(Table 3). For extraction using a MRES of 2:1, the extraction yield increased about 0.53 p.p. comparing the results with ethanol 95% (Run 30) to ethanol 99.8% (Run 20). These results indicate the viability of the use of a lower ethanol purity (for example azeotropic ethanol) in the extraction with scCO2+EtOH in terms of keeping high extraction yields.

ethanol addition of 10.23% in the CXL mixture (MRES = 0.5:1) increased the overall extraction yield from 2.21% to 12.37% in 55 min of extraction. At MRES of 2:1, the highest extraction yield of 15.87% (in 25 min of extraction) was obtained at 80 °C and 20 MPa, the same condition where the highest extraction yield of 15.74% was obtained using pressurized ethanol, but in a extraction time of 55 min. However, as it can be noted, the constant initial extraction rate (CER step) for scCO2+EtOH (2:1) was higher than for PLE extraction with pure ethanol. The influence of the ethanol purity of the solvent (5% water in ethanol) on the extraction yield was evaluated at 60 °C and 15 MPa, using MRES of 0.25:1, 0.5:1, 1:1 and 2:1 and comparing ethanol 95% and 99.8% purity. These results are shown in Fig. 8. The yields obtained with ethanol 99.8% were higher than those obtained with 95% ethanol for all MRES evaluated. However, the yields with ethanol 95% tend to approximate to the results obtained with ethanol 99.8% by increasing the MRES. Increasing the ethanol purity from 95% to 99.8%, for experiments performed with a MRES of 0.5:1, resulted in an extraction yield increase of 7.40% (Run 29) to 9.13% (Run 15), about 1.73 p.p.

3.3.1. Sequential extraction using scCO2+EtOH A sequential extraction was also carried out to evaluate the possibility of enhancing the oil recovery by performing a multistep extraction. The experimental condition selected for this test was 0.5:1 (MRES), 40 °C and 10 MPa (Run 10), because at such condition the extraction yield was 10.34% (around 2/3 of the total extraction capability), but with the highest initial extraction rate and consequently lower extraction time (10 min), and temperature, pressure and lower solvent consumption during dynamic extraction (18.71 g CO2). Additionally, as shown in Fig. 7, the conditions at MRES of 2:1 also presented a high initial extraction rate, such as the extraction at 80 °C and 10 MPa (Run 17), however these extractions used 33.43 g of

Fig. 7. Extraction curves with highest yields for the different solvents scCO2 (40 °C and 20 MPa), scCO2+EtOH at MRES od 0.5:1 (40 °C and 20 MPa), scCO2+EtOH at MRES of 2:1 (80 °C and 20 MPa) and pressurized ethanol (80 °C and 20 MPa). 10

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Fig. 8. Experimental overall extraction curves for spent coffee grounds oil using scCO2+EtOH at 60 °C, 15 MPa and different MRES (0.25:1, 0.5:1, 1:1 and 2:1), using ethanol with 99.8% of purity and ethanol with 95% of purity.

10 MPa was obtained in the extractions using the lowest MRES (0.5:1). As observed in Table 3, the CO2 mass used during the static steps for the sequential extraction was the largest amount among all conditions. Due to limitations of the experimental setup it was needed to stop the extraction, release the CO2 at the end of the CER step to add another charge of ethanol, followed by a new pressurization, which resulted in greater mass of CO2 used. However, this drawback can be easily solved by performing the ethanol addition using a high-pressure pump. Thus, the results presented in this work suggest that the extraction yield of SCG oil can be improved with the addition of ethanol using sequential extraction steps of extraction.

ethanol during the static extraction, while Run 10 used 6.93 g of ethanol. Fig. 9 shows the overall extraction curves at 40 °C and 10 MPa for the sequential extraction and its comparison with others single step extractions with each solvent (scCO2, scCO2+EtOH (0.5:1 and 2:1) and pressurized ethanol). In the sequential extraction, with the accomplishment of a second extraction step, the yield of 10.21% obtained in 10 min of extraction was increased to 13.53% with second extraction step of 12.5 min. As shown in Fig. 10, such enhance obtained using the multistep extraction approached the overall extraction yield to the maximum obtained in this study (single extraction using an ethanol to SCG of 2:1) but using half of the solvent amount. In addition, extraction yield in the sequential approach was higher than the yield obtained for the best experimental condition at MRES of 0.5:1 (40 °C and 20 MPa). It can be notice from Fig. 9 that the increase in the ethanol amount added into the extraction vessel resulted in lower initial rates of extraction. This is due to the increase in mass transfer resistance caused by the ethanol excess inside the extractor vessel in the beginning of the extraction process. Thus, as suggested by the results obtained in this study, the highest initial extraction rate at a fixed condition of 40 °C and

3.4. Fatty acids profile Table 4 shows the fatty acid profile for the SCG oil samples obtained at the highest extraction yields of each solvent (SFE, CXL, PLE and Soxhlet). The major fatty acids found in the SCG oil were linoleic (ranged from 44.05 to 45.79 %) and palmitic (29.70–33.04 %), followed by oleic (8.43–8.89 %) and stearic (7.69–8.79 %). Other fatty acids found were cis-11-eicosenoic, linolenic and tricosanoic. Similar

Fig. 9. Experimental extraction curves for each solvent (scCO2, scCO2+EtOH (MRES = 0.5:1 and 2:1) and pressurized ethanol) at 40 °C and 10 MPa, for sequential extraction at 40 °C and 10 MPa and for scCO2 at 40 °C and 20 MPa. 11

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Fig. 10. Antioxidant activity (AA) by the total phenolic compounds content (TPC) for spent coffee grounds oil.

results of fatty acid profile for SCG oil were found by Akün et al. (2014), using n-hexane, dichloromethane e scCO2 (linoleic 47.77–49.01 %, palmitic 24.47–27.70 %, oleic 9.00–10.39 %, stearic 7.41–8.25 %), and Melo et al. (2014), using n-hexane (linoleic 44.67%, palmitic 37.37%, oleic 8.31%, stearic 7.07%). The fatty acid profile of the SCG oil is similar to cottonseed oil, where the major fatty acid normally found is linoleic (46.7–58.2 %), followed by palmitic (21.4–26, 4%), oleic (14.7–21.7 %) and stearic (2.1–3.3 %) (Knothe, 2002). Table 5 presents the iodine value (IV) for the SCG samples determined by using the fatty acid profile presented in Table 4 The IV results for the different extractions were similar, varying from 90.34 to 93.05 g I2/100 g oil. These values are close to that observed by Najdanovic-Visak et al. (2017) of 83.7 g I2/100 g SCG oil extracted with n-hexane in Soxhlet. Oils with low iodine content such as olive oil (80.3 g I2/100 g oil) are of great interest for use in human diets and in food and cosmetic and pharmaceutical industries. The IV values of the SCG oil obtained in this study were lower than IV values observed for other edible oils, such as sunflower oil (102.02 g I2/100 g oil), peanut oil (119.19 g I2/100 g oil) (Bozdogan Konuskan et al., 2019) and corn oil with (130.6 g I2/100 g oil) (El-Hadad and Tikhomirova, 2018). In

Table 5 Iodine values (IV) and saponification value (SV) for spent coffee grounds (SCG) oil samples. Extraction

Solvent

Condition

Iodine value (g I2/ 100 g oil)

Saponification value (mg KOH/g oil)

CXL

scCO2+EtOH(0.5:1) scCO2+EtOH(0.5:1) scCO2+EtOH(0.5:1) scCO2+EtOH(2:1) scCO2+EtOH(2:1) scCO2+EtOH(2:1)

40 °C/10 MPa 40 °C/20 MPa 60 °C/15 MPa 80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

90.34 92.51 91.12 92.26 92.52 91.37

197.12 196.76 196.98 196.73 196.77 196.27

SFE

scCO2

40 °C/20 MPa

91.58

196.49

PLE

EtOH EtOH EtOH

80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

92.56 93.05 91.23

196.63 196.13 196.56

Soxhlet

n-Hexane EtAC Ethanol

– – –

92.59 92.44 92.22

194.75 197.03 196.90

Table 4 Fatty acid profile for spent coffee grounds (SCG) oil samples. Extraction (Solvent) Condition

Palmitic acid (C16:0)

Stearic acid (C18:0)

Oleic acid (C18:1)

Linoleic acid (C18:2)

Gama- linolenic acid (C18:3) (%)

Cis-11-eicosenoic acid (C20:1)

Docosadienoic acid (C22:2)

Tricosanoic acid (C23:0)

CXL (scCO2+EtOH) 40 °C/10 MPa/0.5:1 40 °C/20 MPa/0.5:1 60 °C/15 MPa/0.5:1 80 °C/10 MPa/2:1 40 °C/20 MPa/2:1 80 °C/20 MPa/2:1

31.53 31.42 31.69 31.41 31.36 31.08

8.79 8.30 8.52 8.42 8.41 8.26

8.88 8.79 8.62 8.57 8.61 8.53

44.05 45.38 44.68 45.35 45.48 44.92

1.52 1.49 1.60 1.38 1.41 1.38

3.22 3.03 3.06 3.03 3.01 2.94

0.95 0.77 0.89 0.99 0.87 2.05

1.06 0.82 0.94 0.85 0.85 0.84

SFE (scCO2) 40 °C/20 MPa

33.04

7.69

8.43

45.38

1.43

2.28

1.21

0.54

PLE (EtOH) 80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

30.43 29.70 30.86

8.46 8.15 8.47

8.66 8.53 8.89

45.39 45.79 44.57

1.68 1.67 1.72

3.21 3.06 3.14

1.22 2.18 1.44

0.95 0.92 0.91

Soxhlet n-hexane EtAc Ethanol

31.31 31.44 31.55

8.60 8.34 8.47

8.65 8.78 8.59

45.43 45.34 45.29

1.36 1.67 1.40

3.17 3.04 3.09

0.57 0.49 0.73

0.91 0.90 0.88

Standard uncertainties estimated for the analysis were considered < 1%. 12

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Table 6 Total phenolic content (TPC) and antioxidant activity (AA) by ABTS and DPPH methods for spent coffee grounds oil. Extraction Solvent

CXL scCO2+EtOH(0.5:1) scCO2+EtOH(0.5:1) scCO2+EtOH(0.5:1) scCO2+EtOH(2:1) scCO2+EtOH(2:1) scCO2+EtOH(2:1) SFE scCO2 PLE EtOH EtOH EtOH Soxhlet n-Hexane EtAc Ethanol #

Experimental condition

TPC mg GAE/100 g oil

AA ABTS μmol TEAC/100 g oil

AA DPPH

40 °C/10 MPa 40 °C/20 MPa 60 °C/15 MPa 80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

269 272 411 ± 79# 393 295 348

4610 3308 4143 ± 115# 4061 3440 3629

3148 2840 3215 ± 383# 2868 2691 2815

40 °C/20 MPa

64

397

345

80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

406 ± 40# 267 351

4559 ± 760# 4451 3905

3534 ± 420# 2841 3199

– – –

66 177 330

676 2443 3502

642 1618 2606

Expanded uncertainties estimated with 95% of confidence level considering triplicate experiments.

regulation established by the EU no. 432/2012, in which the minimum of 25 mg GAE/100 g oil is established (Khmelinskii et al., 2019). In addition, the samples of SCG oil showed TPC higher than the values of 13.8–70.0 mg GAE/100 g oil found in virgin olive oil by Khmelinski et al. (2018).

addition, the IV values for SCG oil obtained in this work are within the European standard for biodiesel production, in which the maximum value of 120 g I2/100 g oil is established (Ramos et al., 2009). Saponification values (SV), in Table 5, presented similar values for all experimental conditions and the solvents used in this study, ranging between 194.75 and 197.12 mg KOH/g oil. Among the solvents analyzed, the lowest SV value was obtained with n-hexane in Soxhlet (194.75 mg KOH/g oil). Values of 173.9 mg KOH/g oil and 183.0 mg KOH/g of SCG oil were obtained by Al-Hamare et al. (2012) using nhexane and ethanol in Soxhlet, respectively. However, SV values of SCG oil obtained in this work were close to the SV for the corn oil (190.3 mg KOH/g oil, El-Hadad e Tikhomirova, 2018).

3.6. Antioxidant by ABTS and DPPH methods Table 6 shows the results for antioxidant activity (AA) for the SCG oil samples with the highest yields for each extraction (SFE, CXL, PLE and Soxhlet), performed according to ABTS and DPPH analyses. In the Soxhlet extraction, an increase in AA values was observed with the increase in the solvent polarity, thus, the highest AA was obtained with ethanol (3501.80 μmol TEAC/100 g oil). The highest AA performed by the ABTS method was 4609.53 μmol TEAC/100 g oil, obtained with scCO2+EtOH (0.5:1) at 40 °C and 10 MPa. The highest AA by the DPPH method was 3534.16 μmol TEAC/100 g oil, obtained with pressurized ethanol at 80 °C and 10 MPa. Difference in the content of compounds responsible for the antioxidant characteristics detected by the DPPH and ABTS method in each sample can generate results like above, where the highest AA were obtained at different samples. Hence, the necessity of performing more than one AA analysis method is evidenced. For the extractions with scCO2+EtOH and pressurized ethanol, in both methods of analysis (ABTS and DPPH), Table 6 shows that AA is improved by increasing temperature and decreasing pressure, a behavior similar to that observed for TPC. The relationship between the values of AA and TPC are shown in Fig. 10, where a linear correlation between these variables with R² of 0.76 for the ABTS and R² method of 0.86 for DPPH methods are observed. Such a relationship between TPC and AA is expected since the antioxidant activity of the SCG oil is attributed to the phenolic compounds present in the oil. The antioxidant activity of phenolic compounds increases the resistance of the oil to oxidation and increases its range of applications (Khmelinskii et al., 2019). In general, it can be observed that at same experimental condition (40 °C and 20 MPa), in Table 6, the addition of ethanol to scCO2 (scCO2+EtOH) enhanced the AA (ABTS and DPPH). Also, through increasing the amount of ethanol in the extractor increased the AA by the ABTS method, and similar results were found for AA by the DPPH method. The use of scCO2+EtOH resulted in AA ABTS values of 3308.30–4609.53 μmol TEAC/100 g oil and AA DPPH of 2691.25–3214.76 μmol TEAC/100 g oil, similar values to those obtained with pressurized ethanol for AA ABTS (3904.57–4559.42 μmol TEAC/100 g oil) and AA DPPH (3199.03–3534.16 μmol TEAC/100 g

3.5. Total Phenolic Content (TPC) Total phenolic content (TPC), presented in Table 6, ranged from 63.90 to 411.10 ± 18.45 mg GAE/100 g oil. The highest TPC value of 411.10 ± 18.45 mg GAE/100 g oil was obtained for the extraction with scCO2+EtOH at 60 °C, 15 MPa and MRES of 0.5:1. In the Soxhlet extraction, TPC values increased through increasing solvent polarity, hence the highest TPC value was obtained with ethanol (329.94 mg GAE/100 g oil) and the lowest value was obtained with n-hexane (66.12 mg GAE/100 g oil). From the results presented in Table 6, at 40 °C and 20 MPa, the lowest TPC value among the solvents (scCO2+EtOH, scCO2 and pressurized ethanol) was 63.90 mg GAE/100 g oil obtained with scCO2. Adding ethanol in the extractor vessel with scCO2 at an ethanol to SCG mass ratio (MRES) 0.5:1 the TPC increased from 63.90 to 272.43 mg GAE/100 g oil, due to the ethanol polarity and consequent high affinity for the analytes. At same condition (40 °C and 20 MPa), with MRES increasing from 0.5:1 to 2:1 in extractions using scCO2+EtOH, the TPC increased from 272.43 to 294.47 mg GAE/100 g oil. Overall, the results indicate that increasing the amount of ethanol in the extractor vessel increases the recovery of phenolic compounds. Thus, TPC values obtained with scCO2+EtOH at MRES = 2:1 (294.47 to 392.96 mg GAE/ 100 g oil) were high and similar to those observed in pressurized ethanol extraction (267.12 to 406. 12 mg GAE/100 g oil), however, in the extractions with scCO2+EtOH (2:1) smaller amounts of ethanol were used. In addition, it can be observed in Table 6 that the extractions with scCO2+EtOH and pressurized ethanol showed similar behavior, in which high temperatures combined with low pressures produced high TPC values for the SCG oil. TPC values for SCG oil (63.90–411.10 ± 18.45 mg GAE/100 g oil) are classified as having human health benefits according to the 13

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Table 7 Profile of phenolic acids and caffeine for spent coffee grounds oil extracted from different methods. Solvent (MRES)

CXL scCO2+EtOH(0.5:1) scCO2+EtOH(0.5:1) scCO2+EtOH(0.5:1) scCO2+EtOH(2:1) scCO2+EtOH(2:1) scCO2+EtOH(2:1) SFE scCO2 PLE EtOH EtOH EtOH Soxhlet n-Hexane AcEt Ethanol

ND: not detected.

#

Experimental condition

Caffeine (mg/100 g oil)

Dihydroxybenzoic acid (mg/100 g oil)

Caffeic acid

p-Coumaric acid

Ferulic acid

40 °C/10 MPa 40 °C/20 MPa 60 °C/15 MPa 80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

421.10 439.15 711.70 ± 45.17# 593.33 404.58 612.71

5.92 5.87 8.81 ± 0.46# 17.66 7.38 16.80

4.68 3.97 5.77 ± 2.08# 9.36 5.47 9.02

2.59 2.10 2.33 ± 0.33# 2.18 2.61 1.60

1.41 1.74 1.76 ± 0.09# 1.54 1.35 1.65

40 °C/20 MPa

64.05

ND

ND

ND

ND

80 °C/10 MPa 40 °C/20 MPa 80 °C/20 MPa

451.05 ± 49.10# 196.36 393.51

13.53 ± 1.42# 3.85 11.2

9.08 ± 1.55# 2.87 7.69

3.44 ± 0.82# 1.25 2.75

1.49 ± 0.34# 0.93 1.34

– – –

23.06 24.94 550.23

ND 4.8 16.94

ND 2.34 7.13

ND 0.24 2.29

ND 1.31 2.68

Average and expanded uncertainties estimated with 95% of confidence level in triplicate experiments.

higher values than those observed in this work. On the other hand, Shang et al. (2016) obtained caffeine content of 30–130 mg/100 g SCG using PLE extraction with ethanol, values similar to those obtained in the extractions of the present study (3–97 mg/ g SCG). These variations may be related to the differences between the coffee blends used in each study, since coffee has different caffeine contents depending on the species (arabica or robust) (Dorsey and Jones, 2017).

oil), but with lower amount of solvent and extraction time. Thus, the scCO2+EtOH extraction is presented as an interesting alternative aiming the spent coffee grounds oil extraction with high antioxidant activities. 3.7. Phenolics compounds and caffeine The phenolic compounds and caffeine identified in the samples of SCG oil with the highest overall extraction yield form the different process (SFE, CXL, PLE and Soxhlet) are presented in Table 7. The phenolic acids were detected in all samples except those obtained with scCO2 and n-hexane (non-polar solvents). The main phenolic acid found was dihydroxybenzoic acid, followed by the caffeic acid. The highest concentrations of dihydroxybenzoic and caffeic acid were 17.66 mg/ 100 g oil and 9.36 mg/100 g oil, respectively, obtained with scCO2+EtOH (2:1) at 80 °C and 10 MPa. In the Soxhlet extraction, an increase in phenolic acid content was observed with increasing polarity and boiling temperature of the solvent. For the extractions using scCO2+EtOH and pressurized ethanol, it is observed in Table 7 that high temperatures and low pressures favor the recovery of phenolic acids. In the extraction with scCO2+EtOH the increase of the MRES resulted in an increase in the phenolic acid content. Also, the values of phenolic acid content obtained with scCO2+EtOH (2:1) were higher than those obtained with pressurized ethanol. The caffeine content in the SCG oil samples, in Table 7, ranged from 23.06 to 711.70 mg/100 g oil. The highest caffeine content (711.70 mg/ 100 g oil) was obtained with scCO2+EtOH (0.5:1) at 60 °C and 15 MPa. Addition of ethanol to scCO2 (40 °C and 20 MPa) raised the caffeine content from 64.05 to 439.15 mg/100 g oil. In the CXL extractions with scCO2+EtOH, the increase in temperature and pressure resulted in higher caffeine contents. While in the PLE extraction with pressurized ethanol, higher contents of caffeine were obtained at higher temperature and lower pressure. The highest average caffeine content was obtained with scCO2+EtOH (2:1). However, at 40 °C and 20 MPa, it was observed that the increase in MRES resulted in a decrease in the caffeine content. Caffeine is considered the most consumed stimulant for the central nervous system, whether as coffee or tea drink, soft drinks or chocolates. In addition, it is widely used as a supporting or agent in pharmaceutical formulations. The consumption of moderate doses has stimulating effect and reduces fatigue, without causing damages (Andrade et al., 2012). The caffeine content obtained by Andrade et al. (2012) in Soxhlet with n-hexane was 210 mg/100 g oil and in the extraction with scCO2 values between 2720 and 4130 mg/100 g oil were obtained,

4. Conclusions In this work, the spent coffee grounds (SCG) oil extraction with scCO2+EtOH as solvent was studied and compared to the extractions with supercritical carbon dioxide (scCO2), pressurized ethanol (EtOH) and to the conventional Soxhlet technique. The results demonstrated the technical feasibility of SCG oil extraction using scCO2+EtOH, where the addition of ethanol significantly enhanced the extraction recovery. Among of the process parameters (temperature, pressure, mass ratio of ethanol to SCG, confinement time and ethanol purity), the mass ratio of ethanol to SCG (MRES) was the most influent in the extraction yield and chemical profile of the oil sample obtained. At MRES of 0.5:1 the highest overall extraction yield was 12.85% obtained at lower temperature (40 °C) and higher pressure (20 MPa), same condition where the highest result for the SFE extraction was obtained. The highest extraction yield obtained among the overall solvents was 15.87% (in 25 min of extraction) obtained with scCO2+EtOH at MRES 2:1, at highest temperature (80 °C) and pressure (20 MPa). The use of scCO2+EtOH allowed the extraction of oil with high yield from 10 to 20 MPa, pressures where low yields and high times of extraction were observed in the SFE with scCO2. The yields of the CXL extraction were comparable to the yields obtained in the PLE but using lower amount of ethanol and the extraction time. The fatty acid profiles of the oils obtained by the different extraction techniques (CXL, SFE, PLE and Soxhlet) were similar. The main fatty acids found in coffee grounds were linoleic (44.05 - 15.79%) and palmitic (29.70–33.04 %). The IV for the SCG oil (90.34–93.05 g I2/100 g oil) were lower than the IV observed for other edible oils (sunflower oil, peanut oil and corn oil) and were within the European standard for biodiesel production. The results of total phenolic content (TPC) and antioxidant activity (AA) for the SCG oil samples obtained with scCO2+EtOH (2:1) were similar to those obtained with pressurized ethanol, which among the overall solvents had the highest values. The extractions with scCO2+EtOH and pressurized ethanol also showed similar behavior, in which high temperatures combined with low pressures produced high 14

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TPC and AA values for the spent coffee grounds oil. The main phenolic acids found were dihydroxybenzoic acid and caffeic acid. In addition, the highest concentrations of phenolic acids and caffeine on average were obtained with scCO2+EtOH (2:1). For the extractions using scCO2+EtOH and pressurized ethanol the recovery of phenolic acids was favored by high temperatures and low pressures. In general, the spent coffee grounds oil obtained scCO2+EtOH (2:1) presented high yield, total phenolic content, antioxidant activity and caffeine content, demonstrating the feasibility of the extraction process using CO2-expanded ethanol (CXL) to obtain oil from spent coffee grounds.

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