Valorization of Spent Coffee Grounds: Comparing Phenolic Content and Antioxidant Activity in Solid-Liquid vs. Subcritical Water Extraction Methods ^†

Abstract

The aim of this work was to extract phenolic compounds (PCs) from spent coffee grounds (SCGs) using two different methods (solid-liquid extraction (SLE) and subcritical water extraction (SWE)) and compare their total phenolic content (TPC) and their antioxidant activity (AA) by 1,1-diphenyl-2 picrylhydrazyl radical (DPPH^•), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^•+) scavenging activities, and ferric reducing antioxidant power (FRAP) assay. The SWE extracts displayed higher TPC values and higher scavenging activity in the DPPH^• and ABTS^•+ assays. The SLE extracts displayed a higher ferric reducing power. These results highlight the impact of the extraction method in PCs extraction and the antioxidant power of the extracts produced.

1. Introduction

Coffee is one of the world’s most popular and consumed beverages, with very large and increasing levels of production and sales worldwide [1]. In the 2021/2022 coffee year, 175.6 million 60 kg bags were produced, an increase of 4.2% in respect to the previous year [2]. Spent coffee grounds (SCGs) are the most abundant waste (45%) generated by the coffee industry [3]. For every ton of raw coffee bean processed, roughly 650 kg of SCGs are produced, and for every kg of instant coffee produced, 2 kg of SCGs are generated. This amounts to 6 million tons of SCGs produced annually [1,2,3].

With a large amount of SCGs being produced, the valorization of these wastes is of great importance. Various applications have been studied, from the production of biofuels (bioethanol, bio-oils, biodiesel, and biogas) [4] to their incorporation into food products [5], to the extraction of several compounds (e.g., phenolic compounds (PCs), polysaccharides, oligosaccharides, monosaccharides, 5-hydroxymethylfurfural, diterpenes and nitrogenous compounds) [2].

In this study, the quantification of total phenolic content (TPC) and antioxidant activity (AA) of SCGs extracts was performed. The SCG extracts were produced by two different methods, solid-liquid extraction (SLE) and subcritical water extraction (SWE). SLE traditionally uses organic solvents, which are expensive, volatile, flammable, and mostly toxic [6]. The use of these solvents produces extracts that, even with high biological activities, have limited utilization for food applications [7,8]. SWE is a technique that eliminates the need for organic solvents, as only water is used. This technique uses water at pressure and temperature levels below its critical point (Tc = 374.15 °C, Pc = 22.1 MPa) [8,9]. By increasing the temperature, the physico-chemical properties of the subcritical water vary dramatically. At higher temperatures, water has a lower dielectric constant, weakening the hydrogen bonds, making water more similar to less-polar solvents, like methanol and ethanol. This allows the solubilization of more polar PCs at low temperature andless polar PCs at high temperature, resulting in higher extraction yields compared to SLE methods [7,8]. Furthermore, SWE typically requires lower extraction times, and possesses higher selectivity, as changes in temperature allow the targeting of compounds based on their polarity [9]. Different studies have shown that the use of SWE results in higher TPC values and enhanced antioxidant activity in DPPH^• scavenging assays of extracts, such as those derived from coffee silverskin, sage by-products, and winery wastes, when compared to conventional extraction methods [10,11,12].

This study provides a direct comparison between SWE and SLE in the extraction of bioactive compounds and their corresponding AA from SCGs. While other studies have explored the use of SWE for extracting bioactive compounds from SCGs, none have directly compared it to conventional extraction methods. This will help in improving the understanding of the influence of the extraction method on the potential application of SCGs residues for valued-added compounds.

2. Materials and Methods

2.1. Samples

SCGs (50:50 Coffea arabica L. and Coffea robusta L. blend) were obtained from MoCoffee Europe (Azambuja, Portugal). SCGs were dried in a dehydrator under 41 °C until they reached less than 10% moisture and stored in the dark until further use.

2.2. Solid-Liquid Extraction (SLE)

The SLEs were performed using stirring maceration extractions at two different conditions: (1) SCG40 °C—biomass/solvent ratio of 1 g:50 mL of 50:50 H₂O:MeOH (v/v), T = 40 °C, t = 1 h; (2) SCG60 °C—ratio of 1 g:100 mL solvent (50:50 H₂O:MeOH (v/v)), T = 60 °C, t = 1 h. These conditions were based on an optimization study performed previously [13]. Afterwards, the extracts were filtered using a paper filter (FILTER-LAB^®, Barcelona, Spain) and the solvent mixture was evaporated in a rotary evaporator. Samples were redissolved in 50:50 H₂O:MeOH (v/v) to a concentration of 10 mg/mL.

2.3. Subcritical Water Extraction (SWE)

The SWEs were performed in a Parr Series 4560 Reactor connected to a Parr 4848 Reactor Controller (Moline, IL, USA) at two different conditions (1) SWE100 °C—biomass/solvent ratio of 2 g:200 mL H₂O, P = 60 bar, T = 100 °C; (2) SWE150 °C—ratio of 2 g:200 mL solvent, P = 60 bar, T = 150 °C. Afterwards, the extracts were filtered using a paper filter (FILTER-LAB^®, Barcelona, Spain) and the samples were lyophilized. Samples were redissolved in H₂O to a concentration of 10 mg/mL.

2.4. Total Phenolic Content

The total phenolic content was evaluated using the Folin–Ciocalteu method, according to Macedo et al. [14], with minor modifications. The calibration curve was constructed using gallic acid solutions between 10 and 200 mg/L. The analysis was performed in triplicate with a microplate reader (Synergy HT, Biotek Instruments, Winooski, VT, USA). The results are expressed as mg gallic acid equivalents (GAE)/g extract dw.

2.5. Antioxidant Activity

The antioxidant activity was measured by performing three assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH^•) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+) scavenging activities, and Ferric Reducing Antioxidant Power (FRAP), according to Macedo et al. [14], with minor modifications. For DPPH^• assay, a calibration curve was established at concentrations between 12.5 and 125 mg/L. An alcoholic DPPH^• solution was prepared by dissolving 1 mg of reagent in 25 mL ethanol. Next, 25 µL of the sample or standard solution was added to each well, to which 200 µL of the DPPH^• solution was added, incubated for 30 min in the dark, and mixed for 30 s, and a measurement was performed at 517 nm. The AA was expressed in mg Trolox equivalents per mass of dry sample (mg TE/g dw). For the ABTS^•+ assay, a calibration curve was established at concentrations between 5 and 100 mg/L. The ABTS^•+ solution was prepared by mixing equal volumes of 7 mM ABTS^•+ and 2.45 mM potassium persulfate, which was left in the dark, at room temperature, for 16 h. The solution was then filtered and diluted to reach an absorbance of 0.700 ± 0.02 at 734 nm. Next, 20 µL of the sample or standard solution was added to each well, along with 180 µL of ABTS^•+ solution, incubated for 6 min, and measured at 734 nm. The AA was expressed in mg TE/g dw.

The FRAP reagent was prepared by mixing an acetate buffer solution (300 mmol/L), TPTZ (10 mmol/L), and FeCl₃ at a 10:1:1 ratio. A calibration curve was established at concentrations between 5 and 100 mg/L. To each well, 20 µL of sample or standard solution and 180 µL of FRAP reagent were added. The solution was incubated at 37 °C for 4 min and the measurement was performed at 593 nm. The reducing power was expressed in mg ascorbic acid equivalents per mass of dry sample (mg AAE/g dw).

All assays were executed in triplicate and were performed using a microplate reader (Synergy HT, Biotek Instruments, Winooski, VT, USA).

2.6. Statistical Analysis

The results are presented as the mean ± standard deviation based on a minimum of three replicates. An ANOVA with Tukey post hoc test was conducted to compare TPC values and antioxidant activities between the extracts using GraphPad Prism (version 8.0.1). Statistical significance was set as p < 0.05.

3. Results

3.1. Total Phenolic Content

The results obtained for the TPC are presented in

Table 1. TPC of the coffee extracts.

Extract	TPC (mg GAE/g dw)
SCG40 °C	134.64 ± 14.73 c
SCG60 °C	104.30 ± 14.56 c
SWE100 °C	170.04 ± 13.16 b
SWE150 °C	331.61 ± 27.85 a

dw—dry weight; GAE—gallic acid equivalents; different superscript lowercase letters correspond to statistically significant differences at p < 0.05.

. The SWE presented a higher TPC, especially SWE150 °C (331.61 ± 27.85 mg GAE/g dw) (p < 0.0001). This demonstrates that SWE is a better extraction method than SLE, and that the use of 150 °C yields better TPC results than 100 °C in the SWE method.

3.2. Antioxidant Activity

The results obtained for the antioxidant activity assays are presented in

Table 2. Antioxidant activity of the coffee extracts.

Extract	DPPH• (mg TE/g dw)	ABTS•+ (mg TE/g dw)	FRAP (mg AAE/g dw)
SCG40 °C	142.09 ± 40.59 c	219.44 ± 26.61 b	87.79 ± 6.65 a
SCG60 °C	120.16 ± 23.80 d	193.32 ± 59.10 b	80.02 ± 15.55 a
SWE100 °C	217.51 ± 15.52 b	975.02 ± 147.80 a	55.82 ± 26.75 b
SWE150 °C	394.17 ± 18.46 a	958.54 ± 184.14 a	42.79 ± 11.90 b

AAE—ascorbic acid equivalents; dw—dry weight; TE—Trolox equivalents; different superscript lowercase letters correspond to statistically significant differences at p < 0.05.

. SWE150 °C displayed the strongest antioxidant activity in the DPPH^• scavenging assay (p < 0.0001), while SWE100 °C displayed a slightly higher result in the ABTS^•+ scavenging assay, with no significant difference to SWE150 °C, but with results vastly superior to the SLE. In the FRAP assay, SCG40 °C displayed the best results, although no significant difference was found with SCG60 °C, and the SWE method displayed the weakest results.

4. Discussion

The use of subcritical water induced higher PC extraction amounts, and increased the AA in the DPPH^• and ABTS^•+ scavenging assays; however, SWE extracts displayed the weakest results in the FRAP assay. The increase in temperature from 100 to 150 °C resulted in a drastic increase in TPC, and DPPH^• scavenging activity, almost doubling in the phenolic content. Andrade et al. [15] produced SCG extracts by different methods, namely, ultrasound-assisted extraction (UAE), Soxhlet, and supercritical fluid extraction (SFE). The highest TPC value obtained was for the UAE (587 ± 46 mg chlorogenic acid equivalent (CAE)/g extract), while the highest value found using a Soxhlet apparatus was 182.6 ± 28.2 mg CAE/g extract, with ethyl acetate as solvent, and for the SFE, a maximum of 57 ± 3 mg CAE/g extract was achieved, with a pressure of 200 bar, 50 °C using CO₂ with 4% ethanol as the co-solvent. Pavlović et al. [16] obtained extracts with a TPC of 398.95 mg GAE/g extract with microwave-assisted extraction (MAE), using a 80:20 H₂O:EtOH (v/v) solution under just 40 s of microwave radiation (80 W). Acevedo et al. [17] utilized 50:50 H₂O:EtOH (v/v) at a solvent/solid ratio of 5:1 and managed to achieve a TPC of 273.34 ± 34.17 mg GAE/g dw. Bouhlal et al. [18] produced extracts from SCGs of a mix of Arabica and Robusta coffee, by a Soxhlet using 25:75 (v/v) H₂O:EtOH and obtained a TPC value of 129.43 ± 6.05 mg GAE/g extract. The differences obtained in different studies might be explained by solvent polarity and extraction method used, and the climatic conditions in which coffee plants were grown, that can influence the quantity of secondary metabolites produced [18,19]. Furthermore, recent studies have shown that the Folin–Ciocalteu reagent reacts with other compounds. The increase in temperature induces Maillard reaction and caramelization reactions, generating many different Maillard reaction products (MRPs), such as melanoidins. These compounds have been shown to possess antioxidant capacities, which can explain the increased AA in SWE [20]. Therefore, the TPC method might not be fully representative of PC levels in extracts [21]. Zengin et al. [21] reported DPPH^• values of 274.44 ± 4.29 and 296.78 ± 7.08 mg TE/g dw in a UAE using 50:50 MeOH:H₂O (v/v) and 70:30 EtOH:H₂O (v/v), respectively. This is significantly higher than the values obtained with the SLEs reported herein. The authors [21] also reported values for ABTS^•+ and FRAP assays, with values of 218.75 ± 6.88 and 276.19 ± 9.65 mg TE/g dw for ABTS^•+ in methanolic and ethanolic extracts, respectively, and 235.47 ± 4.98 and 277.15 ± 3.22 mg TE/g dw for FRAP in the methanolic and ethanolic extracts, respectively. The values reported by these authors are higher than the ones obtained in the SLEs reported here. The results reported by Zengin et al. [21] are, however, significantly lower than the values depicted in Table 2 for the SWE for the ABTS^•+ scavenging activity, despite being higher for the FRAP assay in both methods. Zuorro [22] extracted polyphenols from SCGs, using 1000 mL solvent (57.7% EtOH in H₂O) and 20.8 g of SCG. The obtained extracts displayed antioxidant activity in DPPH^• and ABTS^•+ scavenging assays of 1135 ± 38 and 1583 ± 57 μmol TE/g dw, respectively. Hu et al. [23] obtained ABTS^•+ values of 182.25 ± 5.84 and 198.60 ± 7.23 mg TE/g dw from coffee pulp using SFE with CO₂ and aqueous extractions, respectively. Ballesteros et al. [24] optimized the production of SCG extracts by autohydrolysis (15 mL H₂O/g SCG, 200 °C, 50 min) and obtained FRAP results of 69.50 mg Fe(II)/g SCG. Xu et al. [25] performed SWEs on SCGs and reported that TPC significantly increased as temperature increased, up to 170 °C, after which it decreased. The solid-to-liquid ratio also played a role in the TPC and AA, with an increase in the ratio resulting in lower TPC and AA, possibly due to the decreased yield of phenolic extraction. At optimal conditions, the predicted TPC was 86.23 mg GAE/g, and AA 81.38 mmol TE/100 g and 42.13 mmol TE/100 g for ABTS^•+ and DPPH^•, respectively. The results are lower than the ones reported in this study. Pedras et al. [26] performed a semi-continuous extraction with subcritical water at different temperatures, pressures and water flowrates. A maximum yield of 4 g per 100 g dry SCG was obtained at 200 °C, 70 bar, and 10 mL/min. The maximum TPC value achieved was 155.7 ± 5.6 mg/g extract, and DPPH^• activity was also assessed, with the lowest EC₅₀ obtained being 0.60 ± 0.01 mg/mg DPPH^•. Narita et al. [11] performed SWEs from coffee silverskin and analyzed their AA with two assays (DPPH^• and hydrophilic oxygen radical absorption capacity (H-ORAC)). The AA increased concomitantly with the temperature, up to 270 °C. A high correlation was also found between AA and TPC.

5. Conclusions

In this paper, two different methods were used for the extraction of PCs from SCGs. For the first time, a direct comparison was drawn between an SLE and an SWE, along with the phenolic content and AA of the produced extracts. SCG extracts are a potentially rich source of PCs, with great antioxidant activity. SWE produced extracts with higher TPC values, higher DPPH^• and ABTS^•+ scavenging activities, but lower ferric reducing power than SLE. These results offer valuable information for the future valorization of these by-products. Different extraction methods, under different conditions, yield different results, which can be tailored for specific applications. Further investigation is necessary to optimize these extractions, and to understand the influence of growth conditions of the plants, affecting the production of secondary metabolites.