Optimization of ultrasonic-assisted extraction of kahweol and cafestol from roasted coffee using response surface methodology

LWT - Food Science and Technology 117 (2020) 108593

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Optimization of ultrasonic-assisted extraction of kahweol and cafestol from roasted coffee using response surface methodology

T

Mirelli Bianchina, Hugo Henrique Carline de Limab, Alessandra Maffei Monteiroc, Marta de Toledo Benassia,∗ a

Departamento de Ciência e Tecnologia de Alimentos, Universidade Estadual de Londrina, Paraná, Brazil Departamento de Química, Universidade Estadual de Maringá, Paraná, Brazil c Departamento de Química, Universidade Estadual de Londrina, Paraná, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: Diterpenes Ultrasound extraction Time Solvent consumption

Kahweol and cafestol were extracted by ultrasound-assisted extraction, replacing the conventional liquid: liquid method. Three parameters of extraction were investigated: amplitude level (41, 102. 5 and 164 μm), extraction time (2, 4 and 6 min) and solvent volume (2, 4 and 6 mL). All parameters had a significant effect on the extraction (p < 0.05). Higher amplitude (164 μm) and volume near the central point (4 mL) significantly increased kahweol and cafestol contents. The process time effect was differentiated for each diterpene. Optimum conditions for diterpene simultaneous extraction was chosen: 164 μm, 4 mL of solvent and 6 min-extraction. Scanning electron microscopy images showed that the ultrasound treatment caused a physical modification in the matrix. The kahweol and cafestol contents obtained by sonication were lower compared to the observed for liquid/liquid extraction (13% for kahweol and 3% for cafestol). However, sonication had a positive impact, intensifying and accelerating the process. The use of sonication allowed the extractive process to be simplified, reducing the number of stages (two steps compared to the conventional method), the amount of solvent and the analysis time.

Chemical compounds studied in this article: Kahweol (PubChem CID 114778) Cafestol (PubChem CID 108052)

1. Introduction Coffee, one of the most produced and consumed beans worldwide, contains a complex mixture of components such as proteins, carbohydrates, lipids and bioactive compounds including caffeine, trigonelline, chlorogenic acids and diterpenes that make this product have unique characteristics (Gaascht, Dicato, & Diederich, 2015; Kalschne et al., 2018; Kitzberger, Scholz, Pereira, & Benassi, 2013). Some compounds of lipid fraction, such as triacylglycerols, sterols, and the diterpenes are related to quality and functional properties of the coffee brew (Kalschne et al., 2018). Studies about kahweol and cafestol, specific diterpenes of the coffee lipid fraction matter, showed positive health effects due to antioxidant, anti-inflammatory and hepatoprotective action (Huber et al., 2008; Lee, Chae, & Shim, 2012; Lee, Choi, Jeong & 2007; Martini et al., 2016; Wang, Yoon, Sung, Hur, & Park, 2012). Information regarding their bioactive properties has motivated the extraction and application of kahweol and cafestol and coffee oil, which is rich in these diterpenes, allowing their subsequent use in food and nutraceutical formulations (Martinez-Saez & Castillo, 2019), cosmetic

and pharmaceutical products (Wagemaker, Fernandes, Campos, Rodrigues, & Rijo, 2012). There are also interest by fine chemistry industry, which after purification, can commercialize these compounds as analytical standards due their high commercial value and restricted commercial availability. Since kahweol and cafestol are mainly presented an esterified form in the coffee bean (Speer & Koolling-Speer, 2006), basic hydrolysis or transesterification and subsequent purification are commonly used to obtain the free form (Belandria et al., 2016). Among the analytical methods, we highlight the one proposed by Dias et al. (2010), in which direct hot saponification is followed by three steps of liquid/liquid extraction with methyl tert-butyl ether (MTBE) and cleaning up with water, and that has been widely used (Barbosa, De Melo, Coimbra, Passos, & Silva, 2014; Mori et al., 2016; Scholz et al., 2014; Souza & Benassi, 2012; Zhang, Linforth, & Fisk, 2012). The liquid/liquid extraction is the most critical step of the process, since it requires longer time, use of relatively high solvents volumes, and has to be carried out by a trained analyst. These factors are exacerbated when it is intended to perform diterpenes extraction routinely and in large scale.

* Corresponding author. Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 6, Londrina, PR, Brazil E-mail address: [email protected] (M.d.T. Benassi).

https://doi.org/10.1016/j.lwt.2019.108593 Received 3 December 2018; Received in revised form 1 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Experimental procedure to diterpenes extraction by sonication.

2. Materials and methods

Currently, driven by the green extraction trends, new extraction methods have been developed and applied with success as an alternative to traditional extraction procedures. Ultrasound-assisted extraction (UAE) is a useful and attractive technique to efficiently extract natural products, mainly components of the lipid fraction (Tiwari, 2015). Several classes of food components were widely investigated and extracted using UAE (Albahari et al., 2018; Espada-Bellido et al., 2017; GonzálezCenteno, Comas-Serra, Femenia, Rosselló, & Simal, 2015; Goula, 2013; Hashemi, Michiels, Yousefabad, & Hosseini, 2015; Rezende, Nogueira, & Narain, 2017; Wang et al., 2015). However, for the coffee matrix, UAE was only described for extraction of caffeine (Wang, Chou, Sheu, Jang, & Chen, 2011), phenolic compounds (Al-dhabi, Ponmurugan, & Jeganathan, 2017) and oil (Rocha et al., 2014). There are no reports about the use of sonication for kahweol and cafestol extraction. High-energy sound waves cause the cavitation phenomena, which consists of the formation, growth and, collapse of microbubbles near a solid surface, so UAE can increase solvent penetration, improving diffusion and mass transfer processes and make extraction faster and more efficient (Awad, Moharram, Shaltout, Asker, & Youssef, 2012; Mason, 1997). In addition to reducing the time and increasing yield, UAE can simplify extraction methods due to the automation potential (Chemat et al., 2017; Tiwari, 2015). The cavitation effectiveness and consequently the efficiency of the process depend on the operating conditions, like amplitude, frequency, intensity, extraction time and extractive solvent (Chemat et al., 2017; Manson & Lorimer, 2002). Therefore, it is necessary to use appropriate experimental designs to obtain a maximum yield of the desired products. Response surface methodology (RSM) has seen widely applied to analytical extraction procedures. It allows evaluating the complex interactions between several variables in order to improve and optimize processes (Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008; Candioti, De Zan, Cámara, & Goicoechea, 2014). In RSM, the responses can be graphically represented as a solid surface in a three or two-dimensional space helping visualize interest region and localize the optimum points. Besides the contour surfaces, desirability graphs can also be generated. They allow to find the experimental conditions to reach, simultaneously, the optimal value for all the evaluated variables, including the researcher's priorities during the optimization procedure (Candioti et al., 2014). Considering the interest in the development of alternative technologies and automation in the diterpenes extraction process, this research evaluated the sonication effect on the extraction of kahweol and cafestol from roasted coffee, studying the parameters of amplitude level, extraction time and solvent volume. The process variables were optimized to determine the extraction process yields and the results were compared with those obtained by a reference method (Dias et al., 2010) to evaluate the efficiency of the process.

2.1. Chemicals For saponification reaction, potassium hydroxide (KOH) 85% (Synth, Brazil) and ethanol p.a. (Impex, Varginha, Brazil) were used. For the separation procedure and chromatographic analysis, methyl tert-butyl ether (MTBE) 99.5% (Sigma Aldrich, Steinheim, Germany), acetonitrile HPLC grade (Panreac, Barcelona, Spain), water purification system (Milli-Q, Millipore, Billerica, USA) and standards of kahweol and cafestol > 98% (Axxora, San Diego, USA) certified by Alexis Biochemicals (Lausanne, Switzerland) were used. 2.2. Sample Brazilian commercial roasted arabica coffee beans (Spresso Melitta, Avaré, Brazil) were used. The beans were ground in a grinder (Krups GVX 2 mill, China) to obtain particles with 16 mesh. The moisture (4.95 g 100 g−1) was determined using infrared equipment (OHAUS, Parsippany, USA) at 105 °C for 7 min, to express the data on dry basis. 2.3. Experimental procedures Before the extraction procedure, coffee samples (200 ± 0.1 mg) were saponified in a thermostatic bath (MA 127/BO, Marconi, São Paulo, Brazil) with 2 mL of potassium hydroxide (2.5 mol L−1 in solution 96% ethanol v/v) at 80 °C for 1 h (Dias et al., 2010). 2.3.1. Extraction of kahweol and cafestol by the ultrasound system The procedure for diterpenes extraction was adapted from the method described by Dias et al. (2010) replacing the liquid/liquid step by the sonication extraction. After the saponification procedure, the extraction of the unsaponifiable matter (Fig. 1) was carried out with 2.0 mL of distilled water and a certain volume of methyl tert-butyl ether (MTBE) determined by the experimental design (2, 4 or 6 mL). After agitation on a vortex, samples were sonicated with a 3.2 mm diameter titanium solid tip probe fitted in the ultrasound system (model Q700, QSONICA, Newtown, USA). The ultrasound system converts 700 W and 50/60 Hz AC line power to high-frequency electrical energy (20 kHz), which is then transformed to mechanical vibration, amplified and transmitted by the probe. Samples were subjected to sonication at different amplitude levels and process time, according to the experimental design (Subsection 2.5). A part of the samples (Block 1) was centrifuged (Sovall SS-3, Ivan Sorval, New York, USA) at 12560 ×g at room temperature for 2 min, and the organic phase was collected. For samples that were not centrifuged (Block 2), the organic phase was collected after sonication. Distilled water was added (2 mL) to clean up the 2

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extract. The organic extract was collected (Fig. 1) and the solvent was evaporated in a thermostatic bath at 70 °C.

CCFC is an experimental design method that can estimate the main effect of the factors respecting the limiting conditions of the procedure. It is used when it is not possible to extend the axial points beyond the experimental region defined by the upper and lower limits of each factor (Neter, Kutner, Nachtsheim, & Wasserman, 1996), as observed in this research for the amplitude and solvent volume conditions. The amplitude of 164 μm corresponded to 80% of the probe capacity, and was defined as the upper limit. The use of higher amplitudes could cause material wear and corrosion on the probe. The maximum and the minimum solvent volume were used based on the conventional procedure (Dias et al., 2010). The experimental design chosen allowed operating under these limits. As the volume variation is very small, the experimental space is limited, requiring that the axial points of this factor be fixed on the cube faces at high and low levels (+1 and −1). In order to evaluate the effect of an additional centrifugation process on the phase separation, the experimental design was divided into two blocks. In Block 1, after sonication, samples were submitted to centrifugation; in Block 2, the organic phase was directly collected after sonication (Fig. 1 and Table 1). The results were analyzed using the software Statistica™ Version 7 (Stat. Soft. Inc., Tulsa, USA). A secondorder polynomial model (Eq. (1)) was fitted to evaluate the yield (response variable, Y) as a function of independent variables (X1, X2, X3) and their interactions:

2.3.2. Kahweol and cafestol extraction by a conventional method The extraction was performed as described by Dias et al. (2010). After saponification, a liquid/liquid extraction with 2.0 mL of distilled water and 2.0 mL of MTBE was applied. The samples were mixed on a vortex, centrifuged (3 min at 3000 rpm and room temperature) and the organic phase was collected. This last stage was repeated three times. Water was added (2 mL) to clean up the extract, and the solvent was evaporated in a thermostatic bath at 70 °C. Triplicate extractions were performed. 2.4. Chromatographic analysis Kahweol and cafestol were analyzed by HPLC according to Dias et al. (2010). The analysis was carried out in an HPLC (Shimadzu, Kyoto, Japan), equipped with a quaternary solvent organizer pump and degasser (DGU 20 AS), an autosampler (SIL20HT), DAD UV/Vis detector (SPDM20A). Separations were performed using a Kinetex 2.6 μm C18 column (50 × 4.6 mm) (Phenomenex, Torrance, USA) with an isocratic elution (water: acetonitrile, 60:40 v/v) at a flow rate of 0.4 mL min−1. An injection volume of 0,400 μL and oven temperature at 55 °C were applied. The detection was made at 230 nm for cafestol and 290 nm for kahweol, with a run time up to 30 min. Identification was based on retention time and UV spectra. Quantification was carried out by external standardization using 7 point analytical curves with triplicate measurements (R2 ≥ 0.99, p < 0.001) in a concentration range from 30 to 200 μg mL−1 (Dias et al., 2010; Mori et al., 2016). Diterpenes contents were expressed on a dry weight basis (d.b).

y = B0 + B1 X1 + B2 X2 + B3 X3 + B11 X12 + B22 X22 + B33 X32 + B12 X1 X2 + B13 X1 X3 + B23 X2 X3

(1) Where B0 is the constant coefficient; B1, B2 and B3 are the linear coefficients; B11, B22 and B33 are the quadratic coefficients; B12, B13 and B23 are the interaction linear coefficients of the model. The linear and quadratic effects, their interaction and significance were studied for each compound, and evaluated by analysis of variance (ANOVA) at a 95% significance level. The desirability function was used to define the best condition for the simultaneous extraction of kahweol and cafestol. Kahweol and cafestol contents, determined in optimized extraction conditions using sonication, were compared to that obtained by the reference method (Dias et al., 2010).

2.5. Experimental design and statistical analysis A central composite face-centered design (CCFC) was used (23) to verify the effect of the variables in the extraction of kahweol and cafestol by sonication. Eighteen (18) experimental points including the repetitions of the central point (Table 1) were performed in triplicate in a randomized order. The three independent variables studied were amplitude level (X1): 41, 102. 5 and 164 μm; extraction time (X2): 2, 4 and 6 min and volume of MTBE (X3): 2, 4 and 6 mL, while the dependent variables were the kahweol and cafestol yield.

Table 1 Experimental matrix of the central composite face-centered design (23) for kahweol and cafestol (mg 100 g−1) obtained by ultrasound extraction. Run

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Block

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2

Variables Amplitude level (μm)

Process Time (min)

Solvent Volume (mL)

41 164 41 164 41 164 41 164 102.5 102.5 41 164 102.5 102.5 102.5 102.5 102.5 102.5

2 2 6 6 2 2 6 6 4 4 4 4 2 6 4 4 4 4

2 2 2 2 6 6 6 6 4 4 4 4 4 4 2 6 4 4

Mean (triplicates of the process) ± Standard deviation.

3

Kahweol (mg 100 g−1)

Cafestol (mg 100 g−1)

305 ± 8 522 ± 6 421 ± 20 348 ± 15 344 ± 8 467 ± 18 298 ± 19 497 ± 13 471 ± 15 508 ± 12 446 ± 12 476 ± 3 478 ± 19 551 ± 20 390 ± 8 221 ± 7 455 ± 9 563 ± 16

365 ± 8 366 ± 8 494 ± 17 451 ± 16 390 ± 15 492 ± 15 360 ± 20 493 ± 10 474 ± 13 505 ± 14 487 ± 1 509 ± 1 479 ± 20 550 ± 21 488 ± 7 378 ± 19 494 ± 9 553 ± 14

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process, its use is at the discretion of the analyst.

Table 2 Effects of the amplitude level (X1), extraction time (X2) and volume of MTBE (X3) in kahweol and cafestol obtained by ultrasound extraction. Factors

Mean Amplitude (L) Time (L) Time (Q) Volume (Q) Amplitude (L) * Time (L) Amplitude (L) * Volume (L) Time (L) * Volume (L) Block

Kahweol

Kahweol = 483 + 50.50X1 + 55.23X22

Cafestol

Cafestol = 501 + 23.70X1 + 27.24X2

152.94X32

(2)

28.01X1 X2

70.70X32 + 32.71X1 X3

32.41X2 X3

Effect

p- value

Effect

p- value

(3)

483 101.00 n.s 110.46 −305.88 −56.02 n.s n.s −20.75

< 0.001 < 0.001 > 0.05 0.013 < 0.001 < 0.001 > 0.05 > 0.05 > 0.05

501 47.41 54.47 n.s −141.37 n.s 65.23 −64.82 14.03

< 0.001 < 0.001 < 0.001 > 0.05 < 0.001 > 0.05 < 0.001 < 0.001 > 0.05

The amplitude (linear term) had a positive effect on the extraction of both diterpenes, with a greater influence on kahweol (Eqs. (2) and (3), Table 2, Fig. 2). Thus, within the range studied, the use of higher amplitude level impacted positively in the diterpenes extraction. The amplitude is related to the acoustic cavitation effect (Tiwari, 2015). Increasing the amplitude leads to more intense wave propagation in the liquid, and cavitation bubbles are more frequently created and collapsed (Zhang et al., 2008). This phenomenon causes matrix cellular rupture and increases the contact surface area between solid and liquid. The solvent diffusion and, consequently, the mass transfer rate, were also increased favoring the extractive process (Goula, 2013; Suslick & Price, 1999). In several studies with high power and process intensity, an increase in the extraction yield was reported both for oil extraction as in rapeseed (Sicaire et al., 2016), soybean (Li, Pordesimo, & Weiss, 2004), and pomegranate seed (Goula, 2013), as well as for compounds such as carotenoids (Li, Fabiano-Tixier, Tomao, Cravotto, & Chemat, 2013), anthocyanins and phenolic compounds (Espada-Bellido et al., 2017) and cholesterol (Li, Chen, & Li, 2017). The highest yield extraction efficiency using UAE is mainly attributed to the effects of ultrasound as microjetting and microstreaming (Luque De Castro & Priego-Capote, 2007; Tiwari, 2015). The surface of the coffee without extraction (Fig. 3A) can be compared with structures of the coffee with ultrasound and liquid/liquid extraction (Fig. 3B and C) using SEM images. Fig. 3A shows the structure of untreated coffee is intact and with no pores. After liquid/liquid extraction, the coffee surface was affected, and well-defined pores appeared. On the other hand, the sonicated samples showed different degrees of degradation. The surface showed few irregular pores with undefined shapes, but most were partially destroyed or with cracks. These physical impacts on coffee treated with UAE were in accordance with the detexturation mechanism described by Chemat et al. (2017). In some cases, the cellular rupture was related to higher extraction yield (Abdullah & Koc, 2013; Fu, Zhang, Cheng, Jia, & Zhang, 2014; Zhang et al., 2008). Chemat et al. (2017) described that cavitation cause high shear forces on media, and the implosion of bubbles on the surface, leading to rupture, fragmentation, erosion and structural destruction. These physical effects occur singly or in synergism, and may contribute to an increase in mass transfer and solute transfer rate, associated with the improved UAE performance in extraction. The process time also had a positive effect (p < 0.05) favoring the

ns: not significant at 95% confidence level (p.value > 0.05); L: linear term Q: quadratic term.

2.6. Analysis of the structure by scanning electron microscopy (SEM) The residue obtained by sonication and by the liquid/liquid procedure, as well as the coffee without treatment were dried in an oven at 105 °C for 1 h. The images were obtained by Quanta Scanning Electron Microscope (model QUANTA FEG 250, Thermo Fisher Scientific, Oregon, USA), and the Digital Micrograph program (Gatam Software Team, Pleaston, USA) was used to analyze the images and compare the extraction methods. 3. Results and discussion For the extractions using sonication, kahweol and cafestol contents varied between 221 and 563 mg 100 g−1 and 360–553 mg 100 g−1, respectively. More considerable variability was observed for kahweol data (Table 1). The three independent variables studied, amplitude level (X1), process time (X2) and solvent volume (X3) had significant (p < 0.05) effects on the diterpenes extraction (Table 2, Fig. 2), although the effects were observed in different terms. The corresponding ANOVA tables are available in Supplementary Material (Tables 1S and 2S). The results were adjusted to second- order polynomial equations (Eqs. (2) and (3)), obtaining the coefficient of determination (R2) of 0.69 and 0.79 for kahweol and cafestol, respectively. There was no significance for the block (Table 2, Fig. 2), demonstrating that the additional centrifugation step did not influence the extraction. The use of centrifugation assists in the sedimentation of particulates, facilitating the separation of the organic phase. Although it is an additional step in the

Fig. 2. Pareto chart of effects and interactions of extraction conditions of (A) kahweol and (B) cafestol obtained by UAE using CCFC experimental desing. 4

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Fig. 3. Scanning electron microscopy images of untreated coffee (A), coffee residue after conventional extraction (B) and after UAE (C) (102.5 μm, 4 min, 4 mL).

extraction of kahweol (quadratic term) and cafestol (linear term) (Eqs. (2) and (3), Table 2, Fig. 2B). This behavior suggests that, in general and in the range of the study, longer process time favors the compounds extraction. Ultrasound is known for accelerating the extractive processes compared to conventional techniques (Fu et al., 2014; Heleno et al., 2016; Wang et al 2015). Literature reports that, regardless of the analyte, the ultrasonic extraction presents an optimal process time, which is associated with two main stages (Goula, 2013; Sahin & Samli, 2013; Toma, Vinatoru, Paniwnyk, & Mason, 2001). On the first stage the solvent penetrates into the sample causing the compounds dissolution, while on the second stage the compounds transfer into the solvent by diffusion through the material pores. In addition, as ultrasonic waves are supplied to the system, there is a slow but gradual increase in temperature due to energy transfer to the sample (Santos & Capelo, 2007; Tiwari, 2015). The increase in temperature can also facilitate the solubility and diffusivity of the solvent, accelerating the extraction (Goula, 2013; Zhang et al., 2008). However, for kahweol a negative interaction between amplitude and process time was observed. Thus, for the specific extraction of this compound, it is better to use shorter extraction times to allow the utilization of higher amplitudes (Fig. 4A). It should be noted that during extraction, under the condition of amplitude and process time at the upper level, an increase in the temperature (50 ± 5 °C) was observed. Higher temperature may favor the extraction, but can also lead to the degradation (Sahin & Samli, 2013). Considering the higher heat lability of kahweol, due to the double bond in the chemical structure (Kurzrock & Speer, 2001; Oigman et al., 2012), exposure to high temperatures may have a more significant impact on this diterpene than on cafestol, resulting in a lower extraction efficiency. For cafestol, the most heat-stable compound, the use of higher amplitude level and process time had a positive effect. However, more extended times of extraction would probably be not interesting, since the effect of sonication could decrease. As temperature increases, the number of cavitation bubbles also increases, but the effect of this phenomenon is inhibited by the vapor pressure formed, blocking the collapse of the

bubbles (Patist & Bates, 2008; Shah, Pandit, & Moholkar, 1999). The solvent volume had the most significant effect on the diterpenes extraction (Eqs. (2) and (3), Table 2) among the studied variables. The use of upper and lower levels of solvent volume (6 and 2 mL) was not adequate for kahweol and cafestol extraction, due to the negative quadratic effect of the model. Interactions among solvent volume and the two other variables (process time and amplitude level) also affected the cafestol extraction (Eq. (3), Table 2). To obtain an efficient extraction, solvent volume should be maintained near the center point, applying higher amplitude levels (Fig. 4B) and extensive process time (Fig. 4C). In general, higher yields are obtained when higher solvent to solid ratios are used, as the increased concentration gradient favors mass transfer and diffusion of the analytes (Heleno et al., 2016; Sahin & Samli, 2013; Zhang et al., 2008). However, higher proportions of liquid may additionally cause the dispersion of the ultrasonic energy, reducing the effects of cavitation and consequently lowering the yield (Al-dhabi et al., 2017). Therefore, the choice of the appropriate volume of solvent is essential (Heleno et al., 2016) and for extracting kahweol and cafestol by sonication, the volume of 4 mL was the most efficient. A high global desirability value (0.94) was obtained considering the amplitude level, process time and solvent volume profiles, showing that the three parameters were maximized. Considering the interest in the simultaneous kahweol and cafestol extraction by sonication, the best extraction was at 164 μm during 6 min and solvent volume of 4 mL (Fig. 5). The results of the UAE extraction were compared to the reference method (Dias et al., 2010) that uses liquid/liquid extraction. No differences were observed comparing the chromatographic profile of the two extracts (Fig. 1S). The kahweol and cafestol contents obtained by liquid/liquid extraction were 582 and 553 mg 100 g−1, respectively. For the ultrasound extraction, at optimized conditions, 508 mg of kahweol 100 g−1 and 536 mg of cafestol 100 g−1 were obtained. Comparing to the reference method, a significantly lower yield 13% for kahweol and 3% for cafestol was observed, but the UAE reduce the amount of solvent and

Fig. 4. Effect of interaction of the extracting variable. (A) Response surface for kahweol extraction by sonication. Volume is set to 4 mL. (B) Response surface for cafestol extraction by sonication. Effect of interaction between amplitude and volume with time set in 6 min and (C) volume and time with amplitude set in 164 μm. 5

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Fig. 5. Profiles of the response values and desirability function for kahweol and cafestol extracted by sonication.

Declarations of interest

process time. It is important to emphasize that the time for realizing a conventional extraction depends on the analyst's ability, but the reduction of two stages of solvent extraction obtained using UAE also reduces the possibility of analyst's mistakes, facilitating and accelerating the process. Overall, the use of ultrasonic extraction, combined with other techniques such as the use of microwave for saponification as recently described by Bianchin, Yamashita, and Benassi (2017), could also be a promising tool for isolation of diterpenes on extended scale. Considering the difficulty of diterpene synthesis, the restricted commercial availability and the high price of commercial standards, efficient methodologies for the extraction of these compounds that allow further purification are of great interest.

None. Acknowledgement This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2019.108593. References

4. Conclusion

Abdullah, M., & Koc, A. B. (2013). Oil removal from waste coffee grounds using twophase solvent extraction enhanced with ultrasonication. Renewable Energy, 50, 965–970. Al-dhabi, N. A., Ponmurugan, K., & Jeganathan, P. M. (2017). Development and validation of ultrasound-assisted solid-liquid extraction of phenolic compounds from waste spent coffee grounds. Ultrasonics Sonochemistry, 34, 206–213. Albahari, P., Jug, M., Radic, K., Jurmanovic, S., Brnčić, M., Brnčić, S. R., et al. (2018). Characterization of olive pomace extract obtained by cyclodextrin-enhanced pulsed ultrasound assisted extraction. LWT- Food Science and Technology, 92, 22–31. Awad, T. S., Moharram, H. A., Shaltout, O. E., Asker, D., & Youssef, M. M. (2012). Applications of ultrasound in analysis, processing and quality control of food: A review. Food Research International, 48, 410–427. Barbosa, H. M. A., De Melo, M. M. R., Coimbra, M. A., Passos, C. P., & Silva, C. M. (2014). Optimization of the supercritical fluid coextraction of oil and diterpenes from spent coffee grounds using experimental design and response surface methodology. The Journal of Supercritical Fluids, 85, 165–172. Belandria, V. P., Oliveira, P. M. A., Chartier, A., Rabi, J. A., Oliveira, A.,L., & Bostyn, S. (2016). Pressurized-fluid extraction of cafestol and kahweol diterpenes from green coffee. Innovative Food Science & Emerging Technologies, 37, 145–152. Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, A. (2008). Response

The variables amplitude level, process time and solvent volume significantly influenced the ultrasound extraction, and the best condition for simultaneous kahweol and cafestol extraction was at 164 μm, 4 mL, and 6 min. Under these conditions, the contents of kahweol and cafestol were similar to those obtained by the liquid/liquid extraction, demonstrating the efficiency of sonication process. It was further observed that UAE intensified and accelerated the extraction process by reducing the number of operational steps, the amount of solvent and the process time. The results indicated that UAE is an economic and promising alternative for kahweol and cafestol extraction in roasted coffee.

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