Applications of Deep Eutectic Solvents in the Recovery of Bioactive Compounds from Brewer Spent Grains

ABSTRACT

Brewers’ spent grain is known as the main by-product obtained during the initial stage of brewing, accounting for 85% of the overall solid by-product from the process. This material contains valuable components, including lignocellulose, proteins, lipids, minerals, vitamins, and phenolic compounds. To recover these constituents, various extraction techniques have been used. Deep Eutectic Solvents (DES) are an emerging group of green solvents utilized for the extraction of organic compounds from biomasses. DES is composed of two or more compounds that act as hydrogen bond acceptors and hydrogen bond donors. Choline chloride is mostly used as a hydrogen bond acceptor in combination with several hydrogen bond donors. While many methods can be used to prepare the solvents, heating with continuous stirring at approximately 50–80°C is the most common technique. To improve extraction efficiency, the physicochemical properties of solvent mixtures need to be considered, such as the composition of those solvents, viscosity, polarity, and molar ratio of solvents. Extraction time and temperature, solid-to-liquid ratio, and water content, are also important. This review intends to present information on deep eutectic solvents and their current application in extracting brewers’ spent grain.

KEYWORDS:

• Brewer spent grains
• deep eutectic solvents
• bioactive compounds
• green extraction technique
• waste valorization

Introduction

Beer is one of the most popular alcoholic drinks, hence being one of the largest industries of the world. According to Conway,^[1] the worldwide beer production amounted to 1.86 billion hectoliters in 2021, about 4,000 million hectoliters higher in volume than that of 2020. China was the top producer in the world, manufacturing over 359 million hectoliters, followed by the United States and Brazil. In Europe, Germany was the leading producer, making over 85 million hectoliters.^[2] The 2023–2027 forecasted compound annual growth rate (CAGR) of the global beer market is 5.44%, increasing its market value from USD 610.05 billion to USD 753.98 billion.^[3] This makes the brewery industry of significant economic importance. Concurrent with this growth, waste from the industry also increases. Every 100-L of beer production generates approximately 20 kg wet weight solid waste,^[4] resulting in 35 to 40 million tons of waste per annum.

The major solid by-product derived from the brewing process is brewers’ spent grain (BSG), which is obtained after the mashing step. During this stage, BSG, which is the insoluble part, is separated from the liquid phase or the wort.^[5] BSG accounts for approximately 85% of the total brewery by-products generated.^[6] BSG contains approximately 75% moisture.^[7] Its solid fraction is composed of 30–70% fibrous materials (cellulose, hemicellulose, and lignin), 20–30% protein, and smaller proportions of lipids, ash, starch, and phenolics compounds (Table 1). Due to its high moisture and nutrient content, BSG is susceptible to spoilage by microbial contamination. Being unstable in quality implies a quick disposal of large amounts of waste, which limits its exploitation. Traditionally BSG is employed as animal feed for cattle,^[16] chicken,^[17] lamb,^[18] and fish.^[19] A number of studies have investigated BSG upcycling in various ways. It was used as a functional ingredient or nutrient-fortifying agent in food products, such as pasta,^[20,21] bread,^[22] muffins,^[23,24] and cookies.^[12] For non-food applications, it was used for biogas production,^[25,26] biochar,^[27] and soil remediation.^[28]

Table 1. Composition of brewing residues reported in recent studies.

Sample	Composition (%w/w dry basis)							Total phenolic compounds (mg GAE/g dry weight)	Reference
	Cellulose	Hemicellulose	Lignin		Protein	Lipid	Ash
			Insoluble	Soluble
BSG	14.0 ± 0.2	32.0 ± 0.6	20.8 ± 0.2		22.1 ± 0.5	6.2 ± 0.3	3.32 ± 0.06	0.365^a, 0.109^b	Alonso-Riaño et al.^[Citation8]
BSG	12.31 ± 1.52	26.28 ± 0.69	3.48 ± 0.43		17.87 ± 1.34	6.72 ± 0.05	2.33 ± 1.17	-	Silva et al.^[Citation9]
craft BSG	38.1 ± 0.2^c				18.7 ± 0.1	6.3 ± 0.01	3.2 ± 0.1	0.23	Jin et al.^[Citation10]
craft BSG	18 ± 2	26 ± 2	13.5 ± 0.5	4.30 ± 0.06	17.7 ± 0.1	5.90 ± 0.01	2.90 ± 0.02	2.72 ± 0.04^a, 1.6 ± 0.2^b	Alonso-Riaño et al.^[Citation11]
BSG	14.73^d				24.83	6.60	3.25	2.33^e	Farcas et al.^[Citation12]
BSG	15.2 ± 0.50	25.1 ± 0.70	7.0 ± 0.50	5.5 ± 0.30	21.2 ± 0.20	-	2.3 ± 0.10	-	Rojas-Chamorro et al.^[Citation13]
Malt bagasse	19.62 ± 0.36^d				23.48 ± 0.63	6.31 ± 0.64	4.30 ± 0.02	1.79 ± 0.03	Saraiva et al.^[Citation14]
Light BSG	-	-	1.52 ± 0.01		20.12 ± 0.1	4.22 ± 0.14	3.23 ± 0.06	0.83 ± 0.01	Kumari et al.^[Citation15]
Dark BSG	-	-	2.54 ± 0.25		22.69 ± 0.07	6.75 ± 0.98	1.97 ± 0.12	4.88 ± 0.59

GAE: gallic acid equivalent, ^aWater extractable polyphenols, ^bEthanol extractable polyphenols, ^creported as total dietary fiber, ^dreported as crude fiber, ^emg GAE/g fresh weight.

Apart from the aforementioned direct uses in food applications, BSG has also been used as a raw material for recovery and isolation of high-value components as highlighted in a number of recent reviews.^[29–31] In recent years, many studies reported the use of deep eutectic solvents (DESs) to replace organic solvents in the extractions of bioactive compounds or organic compounds from various food materials.^[32–34] This review aims to provide information on DESs and the current state of knowledge of their use in the extraction of bioactive compounds from BSG.

BSG composition

Cell wall components

The cell wall components of BSG are mainly the association of polysaccharide polymers such as cellulose and hemicellulose surrounded by lignin sheath,^[31] which account for 30–70% (w/w). Hemicellulose is the major polymeric fraction found in BSG, contributing to 20–40% of the lignocellulosic composition.^[35] The primary component of BSG hemicellulose is arabinoxylan, a heterogeneous polymer formed by β(1→4)-D-xylose and α-L-arabinose. Due to its resistance to enzyme hydrolysis of the digestive tract, arabinoxylan is considered dietary fiber, and may have immunomodulatory properties.^[31] Cellulose and lignin are comparable in their average proportions, which are 15–25% and 10–20%, respectively.^[35] Cellulose is a linear polymer chain comprising of hundreds to thousands units of glucose bound together with β(1→4) glycosidic linkages.^[36] Lignin is an organic polymer that contains phenylpropanoid units including p-coumaryl, coniferyl, and sinapyl alcohols. The heterogeneous branching of lignin is more complex than that of hemicellulose and may have a wide range of degrees of polymerization. Beyond its enzymatic degradation resistance, lignin particularly resists microbial attack due to the toxicity of lignin derivatives and the non-specific adsorption and binding of hydrolytic enzymes.^[37]

Proteins

BSG can be used as an alternative protein source as it contains approximately 15–30% protein.^[35] Being protein-rich, BSG might be used as a raw material for high-protein plant-based products. The main protein components in barley are hordeins (also known as prolamins) and glutelins, accounting for 35–55% and 35–45% of the total protein, respectively.^[29] The rest of the minor protein fractions are albumins and globulins.^[38] It has been reported that BSG also contains many amino acids, both essential (lysine, leucine, histidine, phenylalanine, valine) and non-essential (glutamic acid, aspartic acid, arginine, alanine, serine).^[39,40] During the brewing process, the structures of proteins in barley grain are altered. Details on their structural rearrangement have been discussed in a recent review.^[38]

Lipids

BSG chemical composition is normally high in fibers and proteins; however, it also contains a small amount of lipids (3–10%). del Río et al.^[41] used gas chromatography (GC) and gas chromatography – mass spectrometry (GC – MS) techniques to identify lipid composition in BSG. The authors reported that triglycerides are the most abundant lipid fraction (67%), followed by free fatty acids (18%), diglycerides (7.7%), and monoglycerides (1.6%). Steroid compounds, including steroid hydrocarbons, steroid ketones, free sterols, sterol esters, and sterol glycosides, were also found.^[41] Another study by Lordan et al.^[42] indicated that linoleic (18:2), palmitic (16:0), and oleic (18:1) acids are the main fatty acids detected in BSG.

Phenolic compounds

Phenolic compounds (PCs) are phytochemicals exhibiting bioactivities, such as antimicrobial, antioxidant, anti-inflammatory, and antiproliferative properties.^[40] These groups of compounds are associated with the cell wall components.^[43] Due to this, the presence of husk, pericarp, and seed coat in BSG makes it a good source of PCs (approximately 1–2%).^[35,44] Zuorro et al.^[45] explained that during barley seed development, phenolics are accumulated, especially in the grain husk, hence the high level of PCs in BSG. Ferulic (1860–1948 μg/g) and p-coumaric (565–794 μg/g) acids are the major phenolics in BSG, followed by sinapic, syringic, and caffeic acids.^[46] The level of phenolic compounds is influenced by many pre- and post-harvest factors, such as barley grain species, harvesting time, pedoclimates, and brewing process parameters.^[47]

A comprehensive review of the chemical compositions of BSG can be found in many recent publications.^{[29,38,48,49]} Recent research articles on the physicochemical composition of BSGs obtained from local craft breweries, including malted barley, chocolate malt with malted barley and oat, and malted barley with spelt and rice hulls, was reported by Jin et al.^[10] While BSGs from 8 brewery industries in Poland, Germany, and Estonia (Foundation II German Pale Ale, Bojanowo, WB, Onnevalemi Komonendid, Drunken soiler India pale ale, Sto Mostow, Fest, and Elveliksiirid) were studied by Naibaho and Korzeniowska^[50]

Food applications of BSG

Several studies have investigated alternative uses of BSG due to its contents of nutrition and properties. Comprehensive reviews on the BSG applications in various industries are summarized by Bachmann et al.,^[51] Chetrariu and Dabija,^[52] Emmanuel et al.,^[53] Hejna,^[54] Jackowski et al.,^[30] Karlsen and Skov,^[55] Macias-Garbett et al.,^[56] and Pabbathi et al..^[57] The following section discusses recent progress on the applications of BSG in food products.

The effect of BSG and fermented BSG (FBSG) on the functional and nutritional properties of semolina-based pasta was investigated.^[58] The inclusion of BSG and FBSG enhanced a stronger gluten network formation than the wholemeal pasta since germ and bran particles in wholemeal flour interrupted the formation of the gluten network. There was no significant difference (p > 0.05) observed in the tensile strength between BSG pasta (0.27 ± 0.03) and the semolina control (0.29 ± 0.03). While the firmness of pasta with BSG (2.27 ± 0.40) and FBSG (2.54 ± 0.38) after cooking showed a slight increase compared to the semolina control (2.17 ± 0.37). This might be attributed to the strong gluten network and the increase of protein content from ingredients (BSG and FBSG). Furthermore, fortification of fermented BSG in high content was found to lower the predicted glycaemic index than control pasta. Using einkorn and tritordeum BSGs as functional ingredients in durum wheat pasta was also studied.^[20] Macaroni pasta was produced with 5% and 10% micronized BSG. BSG-enriched pasta showed an improvement in the nutritional composition compared to control pasta, including protein, ash, total dietary fiber, and β-glucan contents, and total antioxidant capacity. The dietary fiber of BSG pasta was significantly higher than that of control (p < 0.05), which could be referred to as “sources of fiber” according to Regulation (EC) number 1924/2006 as containing more than 3 g fiber per 100 g.^[59] The sensory evaluation indicated that pasta with 10% einkorn BSG received high scores in both firmness and global sensorial judgment, while replacement with 10% tritordeum BSG showed the lowest scores. This might depend on the composition of the fiber source.

In order to replace fat in hamburgers, brewing waste (malt bagasse; MB) was added, and the effect on the physicochemical properties of hamburgers was investigated.^[14] Different proportions of MB were used for porcine fat replacement (1–3%). The results revealed that the nutritional value of hamburgers was improved by using MB since it significantly lowered the amount of fat and calories while increasing the amount of protein and antioxidant activity (p < 0.05). The hardness and gumminess of hamburgers were also increased. Differences in sensory profiles between all formulations were not perceived and all the samples were accepted by consumers.

A new chip product with the inclusion of BSG to improve the nutritional values, such as fiber, protein, and antioxidants, was developed by Garrett et al.^[7] Reformulated chips were produced with five different levels of BSG, namely 8%, 18%, 24%, 32%, and 40% (w/w), and the physical and sensory properties of the chips were investigated. The water activity for BSG chips decreased with an increase in BSG level, which may be due to the particle size and water-holding capacity of BSG. Chips showed no differences (p > 0.05) for lightness (L*) and redness (a*), but there were some differences in yellowness (b*). The fracture force, which represents the texture of chips, were not differences (p > 0.05), even though a trend was observed among the samples. No significant differences in flavor were detected, proving that the addition of BSG did not affect the flavor. High level of BSG addition improved the texture rating and willingness to buy by the panelists. Barley rootlets are also used as nutrient enhancer. Up to 25% (w/w) of barley rootlets were incorporated into biscuit formulations, and the sensory effects related to their addition, particularly on the composition of volatile compounds, were investigated.^[60] The higher level of barley rootlets inclusion the higher intensities of the color, odor, flavor, and taste of the biscuits. Volatile compounds, such as 3-methylbutan-1-ol, 2-methylbutan-1-ol, and pentan-1-ol, were the major aroma compounds found in barley rootlets. Barley rootlets imparted an intense note of alcohol or whiskey in the biscuits detected by the panelists. This may be due to the presence of fatty acids in barley rootlets or the processing of barley into malt, such as steeping and germination, which can generate volatile compounds. In terms of overall acceptability and texture, the panelists preferred the biscuits that contained 15% barley rootlets.

The inclusion of BSG flour into muffins was evaluated on its physicochemical properties, bioactive compounds, and sensory quality.^[24] Three different concentrations of BSG (10, 15, and 20 g/100 g flour) were substituted in the muffin formulations. Fortified muffin with 15% BSG exhibited a higher content of protein (13.11 g/100 g dry matter), total dietary fiber (16.88 g/100 g dry matter), and bioactive compounds, including total phenolic content (0.49 ± 0.01 mg GAE/g dry matter) and radical scavenging activity (0.15 ± 0.05 mg ascorbic acid equivalents/g dry matter), compared to control muffins (p < 0.05). All BSG muffins showed a lower L* value than unfortified samples (p < 0.05). It was observed that when BSG content increases, muffins get darker due to the concentration of brown pigment in BSG. Evaluation of sensory quality with hedonic ratings revealed no significant difference in all factors (overall acceptability, appearance, color, taste, and texture) (p > 0.05), whereas incorporated 15% of BSG showed a marginally higher score of color and appearance over control, indicating that BSG muffins might be favorable for consumers.

A study of the effects of BSG (0, 10, 20, and 30% w/w) substitute wheat flour in cookies was reported by Heredia-Sandoval et al.^[61] The cookies were evaluated for phenolic acids, antioxidant capacity, arabinoxylan content, and proximate composition. With the increase in BSG level, the protein, arabinoxylan, and phenolic acid contents were significantly increased (p < 0.05). For overall acceptability, there were no differences (p > 0.05) between control and cookies with up to 20% of BSG substitution. However, the inclusion of 30% BSG in cookies was unacceptable due to its bitter taste and brittle texture. The effect of BSG and fermented BSG was studied on dough quality, bread characteristics, and nutritional value.^[22] The GlutoPeak analysis revealed that high amounts of BSG and FBSG incorporation led to a stronger gluten network in the bread dough, which developed at a faster rate than control (baker’s flour). This indicated that the level of inclusion had a significant impact on the strength of the gluten network (p < 0.001) and the development time (p < 0.001). Additionally, the composition of BSG and FBSG flour, such as fiber (42.6% and 49.4%, respectively), protein (31.4% and 32.4%, respectively), and mineral (3.7%) contents, can also affect the formation of the gluten network. Replacement with fermented BSG enhanced the characteristics of bread by leading to a higher specific volume, reduced crumb hardness, and an extended shelf life during storage. It also reduced the release of sugar through in vitro starch digestion, which positively impacted the nutritional value of bread. Table 2 summarizes the improvement of some food product applications with brewing by-products.

Table 2. The improvement of food products application with brewing by-products.

Raw material	Food products	Improvement of nutritional and sensory properties	Reference
BSG	Cookie	Substitution of BSG improved the protein, arabinoxylan, and phenolic acid contents. The added BSG increased the antioxidant activity (ABTS and DPPH) of the cookies (p < .05). The cookies produced with 10–20% BSG inclusion presented a high overall acceptability score similar to the control cookies, which were assumed to be acceptable.	Heredia-Sandoval et al.^[Citation61]
	Muffin	Fortified muffins with BSG enhanced the chemical composition of final products including protein and total dietary fiber contents. Total phenolic content and free radical scavenging activity were also increased compared to control. The addition of 15% BSG was observed to retain the brightness of muffins and presented a slightly higher mean score for appearance and color.	Shih et al.[24]
	Chip	New chip products containing different levels of BSG showed no significant difference in texture, color (L* and a* values), and flavor. Consumers have preferred the texture of 40% BSG substitution over 8%.	Garrett et al.[7]
	Pasta	The addition of BSG and fermented BSG can improve the nutritional profile of pasta; having higher protein and fiber contents than pasta without BSG. The strong gluten network formation was enhanced with BSG and the pasta firmness was slightly increased.	Neylon et al.[58]
Malt bagasse	Hamburger	The replacement of fat in hamburgers with malt bagasse (MB) increased protein and fiber contents, while reduced fat content. The antioxidant activity (ABTS) was increased according to the increase of MB (p < .5). The inclusion of MB had no influence on the color of hamburgers in terms of lightness and yellowness, the difference between control and hamburger with MB was not detected by consumers.	Saraiva et al.^[Citation14]
Spent malt rootlet	Biscuit	The addition of spent malt rootlets flour (MRF) in biscuits increased the content of polyunsaturated fatty acids (PUFAs) significantly (p < .5) and linoleic acid was the most represented. Monounsaturated fatty acids (MUFAs) content was highest in biscuits with 25% MRF. The presence of limonene was detected according to the amount of MRF added. The biscuit with 15% MRF was favored by the panelists in terms of texture and overall acceptability.	Chiş et al.^[Citation60]

Deep eutectic solvent

Deep eutectic solvent (DES) has emerged since 2003 as a new class of green technologies.^[62] It is commonly defined as a subclass of ionic liquid (IL). DESs are prepared by mixing two or more compounds at certain molar ratios to obtain clear liquid mixtures.^[63] In a binary DES system, one compound acts as hydrogen bond acceptor (HBA) and another compound acts as hydrogen bond donor (HBD). Upon mixing, hydrogen bonds are formed between HBA and HBD, turning the mixture into a homogeneous solution. The resulting solution is called ‘deep eutectic’ solvent because it has a melting point much lower than that of either the individual components.^[64] DESs have some advantageous characteristics over organic solvents traditionally used in extraction of organic compounds, such as low toxicity, cheap material cost, easy handling, and high biodegradability.^[63,64] When compared to ILs, DESs share some physicochemical properties, such as non-flammability, high thermal and electrochemical stability, low volatility, high viscosity, and dissolving capacity (Table 3).^[63,65,66] The physicochemical characteristics of DESs depend on the nature of HBA and HBD. Therefore, it is possible to manipulate DESs’ biological and physicochemical properties to fit a specific application by changing the types of HBA and HBD, their molar ratios, as well as extraction parameters.^[67]

Table 3. Comparative properties of ILs and DESs.^[161,162].

	Ionic liquids (ILs)	Deep eutectic solvents (DESs)
Chemical component	Mixtures of organic heterocyclic cations with organic or inorganic anions.	Mixture of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs).
Intermolecular interaction	Ionic interaction.	Hydrogen bond.
Melting point	Both ILs and DESs exhibit low melting points below 100°C. DESs, however, have significantly lower melting points than each individual component.
Density and viscosity	The density and viscosity of the solvents are influenced by their composition and temperature.
Polarity	Polar	Can be polar (hydrophilic) or non-polar (hydrophobic) depending on the properties of the parent components
toxicity	Less biodegradable, and higher toxicity than DESs.	Stable, biocompatible, more biodegradable, and have lower toxicity than ILs.

Types of DESs

According to Abbott et al.,^[62] DESs can be grouped into the following four types: (1) quaternary ammonium salt (QAS) and metal salt, (2) QAS and metal salt hydrate, (3) QAS and HBD, and (4) metal salt and HBD.

Type (1) is composed of metal salt (SnCl₂, ZnCl₂ or FeCl₃) combined with an organic salt, particularly with a quaternary ammonium salt.^[68] Type (2) is formed by mixing hydrated metal salt with choline chloride (ChCl) or a quaternary ammonium salt.^[69] Type (3) is the most common. It is synthesized by a combination of a quaternary ammonium salt as HBA and either an amide, an alcohol, or a carboxylic acid as a HBD.^[62,70] Type (4) contains a metal salt such as ZnCl_2, instead of organic salt, with a HBD such as urea, ethylene glycol, and acetamide. Recently, a type (5) has been introduced, prepared by using only non-ionic precursors. They are characterized by strong negative deviations from thermodynamic ideality.^[71,72]

Preparation of DESs for extraction of organic compounds from food materials

The performance of a DES system largely depends on its efficiency in dissolving the extractants, therefore the choice of HBA and HBD is crucial. In all applications of hydrophilic DES systems (i.e. extraction of lignocellulosic fractions, proteins, phenolic compounds), ChCl is the most commonly used HBA in research studies.^[34,73,74] A recent review on the extraction of phenolic compounds from natural sources using DES pointed out that 140 out of the total 173 systems reported in research articles published between 2015 and 2021 used ammonium salt as HBA. ChCl and betaine accounted for 75% and 25% of those 140 systems, respectively.^[75] Depending on the application, other HBAs investigated apart from ChCl included acetylcholine chloride, ethylammonium chloride, thymol, menthol, L-carnitine.^[34,73] In the applications of hydrophobic DES systems (for the extraction of neutral and non-polar lipids), although ChCl is workable as a HBA, medium- to long-chain fatty acids (C8-C18), menthol, thymol, tetrabutylammonium chloride, tetraoctylammonium chloride are more adopted as HBA.^[76–78]

Despite the fact that HBA affects the dissolution efficiency, some studies demonstrated that the choice of HBD is the deciding factor for the performance of the DES system employed.^[79,80] The most common HBDs used in hydrophilic DES systems are alcohols, organic acids, sugars, and amides.^[81] Some of the individual compounds, include lactic acid, glucose, glycerol, sucrose, and citric acid have been mostly used.^[75] Recently, the term ‘natural deep eutectic solvent’ (NADES) has been introduced. NADES refers to DES that are particularly prepared from primary metabolites found in living cells, e.g., urea, amino acids, sugars and choline.^[82] A comprehensive list of compounds used as HBA and HBD to prepare DES systems can be found in recent reviews.^{[77,81,83–85]}

Different methods can be used to prepare DES/NADES. The common technique reported in most research studies is heating at approximately 50–80°C with continuous stirring.^[86–88] Other approaches, such as evaporation, microwave-assisted heating, ultrasound-assisted mixing, and twin-screw extrusion, have also been used. A study on ZnCl₂-based DESs with three different HBDs, including glucose, fructose, and lactic acid, were successfully prepared using the evaporation method at 50°C due to their low freezing points and viscosity.^[89] Zhang et al.^[88] investigated the effects of temperatures and molar ratios of ChCl-carboxylic acid DESs prepared by various methods (heating, rotary evaporation, and freeze-drying). The authors suggested that heating is appropriate for liquid acids, while freeze-drying and evaporation are suitable for DES with more carboxyl or hydroxyl groups as HBD. Emerging technologies, such as microwave- and ultrasound-assisted, were also used to prepare ChCl-based NADES with malic acid, urea, and fructose for the extraction of polyphenols from sour cherry pomace. Popovic et al. demonstrated that microwave-assisted heating (180 W) required less than 30 second to obtain a DES solution, while ultrasound-assisted preparation (50°C, 37 kHz) required 6–25 min. The longest time required to yield a transparent liquid was with the conventional method (30–45 min with 50°C heating and 650 rpm stirring).^[90] A new approach for the synthesis of DESs using a twin screw extrusion as a continuous preparation process were explored.^[91] Choline chloride-based DESs with urea, D-fructose, and ZnCl₂ were prepared in a molar ratio of 1:2 with barrel temperatures of 25–85°C and screw speeds of 30–250 rpm. The obtained DES mixtures were colorless, while conventional heating produced brown liquid. This study demonstrated a decrease in preparation times ranged from 30 min to 2 h with a kg-scale of components, making it feasible for a large-scale DES preparation in the food and polymer industries.

Generally, DESs or NADESs are prepared at a high temperature, but not above 100°C.^[92] The preparation can also be achieved by non-thermal techniques. Lyophilization was employed to synthesize the NADES for anthocyanin extraction from the Chilean native species (Luma chequen). Glycerol and glucose as HBD with lactic acid, tartaric acid, and glycerol as HBA were prepared at −40°C under vacuum 0.006 millibar for 18–24 h.^[93] Another study successfully prepared a ternary DES (glycerol:L-proline:sucrose, 9:4:1) by freeze drying which was used for the extraction of ginsenosides from white ginseng.^[94] Apart from lyophilization, mechanical approaches, i.e. grinding, was also investigated.^[95] The ¹H NMR spectra of ChCl-based DESs showed that the DESs obtained from the grinding method were pure, whereas those obtained from the heating method showed impurities ranging from 5–30%. It was similar to the ¹H NMR spectra of DESs reported by Francisco et al.,^[96] however, the authors did not investigate this phenomenon. More details of DES or NADES preparations for bioactive compound extraction are summarized in Table 4.

Table 4. Preparation of DES or NADES for bioactive compound extraction.

Sample	Target compound	DES/NADES			Water content	Solid:Liquid	Preperation method	Extraction method	Result	Reference
		HBA	HBD	Molar ratio
Cistus creticus	Polyphenolic and volatile compounds	trisodium citrate	Glycerol	1:15	20% (v/v)	1 g:30 mL	Stirred (400 rpm)at 70℃	Conventional method: stirred (500 rpm) at 50℃ for 3 h	TPC extraction was achieved by using DES (ChCl:glycerol) with PEF pre-treatment.	Palaiogiannis et al.^[Citation87]
		ChCl	Glycerol	1:2
		glycine	Lactic acid	1:5				PEF (duration 1000 µs/sample, frequency 1 hz,)
		sodium butyrate	Glycerol	1:6
Sour cherry pomace	Polyphenols	ChCl	Malic acid	1:1	20% (w/w)	300 mg:4 mL	Stirred (650 rpm)at 50℃	Conventional method: stirred (650 rpm) at 40℃ for 30 min	DES based on ChCl:malic acid showed high efficiency for anthocyanin extraction (62.33%).	Popovic et al.^[Citation90]
			Urea					UAE at 40℃ for 30 min
			Fructose					MAE at 90W up to 15s
		ChCl	Malic acid	1:1			Using ultrasound in an ultrasonic bath at 50℃, 37 khz, 30 W	Conventional method: stirred (650 rpm) at 40℃ for 30 min
			Urea					UAE at 40℃ for 30 min
			Fructose					MAE at 90W up to 15s
		ChCl	Malic acid	1:1			Microwave irradiation using microwave oven at 180 W within 30s	Conventional method: stirred (650 rpm) at 40℃ for 30 min
			Urea					UAE at 40℃ for 30 min
			Fructose					MAE at 90 W up to 15s
Spent hops	Xanthohumol	ChCl	Glycerol	1:2	5% (w/w)	1:10, 1:20, 1:40, and 1:50 (w/w)	Stirred in an oil bath at 80°C for 20-40 min	Stirred (500 rpm) at 60°C for 1 h in an oil bath	The highest xanthohumol yield (2.30 mg/g spent hop) was obtained using ChCl:propylene glycol (1:2) with 5%wt water, solid:liquid 1:50 (w/w), and 3:1 antisolvent/DES (v/w) at 60 °C for 1 h.	Grudniewska andPopłoński^[Citation97]
			Ethylene glycol		10% (w/w)
			Propylene glycol		5% (w/w)			Extracted with methanol, acetone; stirred (500 rpm) at room temperature for 1 h
			Lactic acid		5% (w/w)
Tartary buckwheat	Rutin	ChCl	1,2-propanediol	1:1	0-50% (w/w)	40 mg:1 mL	Stirred at an elevated temperature(⩽100 °C)	UAE at 40℃ for 1 h, 20 khz, 200 W	ChCl:glucerol showed the highest extraction yield of rutin, 9.6 mg/g (at 20% water, w/w).	Huang et al.^[Citation98]
			Glycerol
			Glucose	2:5
			Sucrose	1:1
			Xylitol	1:2
			Sorbitol	2:5
		Glycerol	L-proline	3:1
			L-alanine
			Glycine
			L-histidine
			L-threonine
			L-lysine	4.5:1
			L-arginine	4.5:2
White ginseng	Ginsenosides	Glycerol: L-proline: Sucrose		9:4:1	added small amount of water	100 mg:1.9 mL	Freeze dried for 24 h or until the mixture reached a constant weight.	UAE for 30 min	DES based on glycerol: L-proline: sucrose in combination with UAE has been shown to be significantly more efficient in a short extraction time (30 min).	Jeong et al.^[Citation94]
								Heated at 70 °C for 1 h
								HRE at 60℃ for 1 h
Olive leaves	Phenolic compounds	ChCl	Acetic acid	1:2	20% (v/v)	0.036 g:1.8 mL	Submerged at 60–80℃ in a water bath, then stirred at 600 rpm	Stirred (800 rpm) at 50℃ for 3 h	ChCl:acetic acid (54.1°C, 50.0% water addition) extracted 15% more phenolic compounds than ethanol.	de Almeida Ponteset al.^[Citation99]
			Malonic acid	1:1
			Malic acid	1:1
			Citric acid	2:1
Foxtail millet bran	Phenolic compounds	ChCl	Oxalic acid	1:1	30% (v/v)	1 g:20 mL	Magnetically stirred at 80℃	UAE at 50℃ for 30 min, 250 W	-The optimized RSM conditions were ultrasonic power at 247 W for 31 min, 29% (v/v) water, and a temperature of 61°C. -Betaine:glycerol and glycerol:citric acid showed the highest TPC.	Zheng et al.^[Citation100]
			Lactic acid	1:2
			Glycerol	1:3
		L-Proline	Glycerol	2:5
			Lactic acid	1:1
		Glycine	L-lactic acid	1:5
		Betaine	Glycerol	1:2
		Glycerol	Citric acid	1:2
			Sodium acetate	3:1
Moringa oleifera L. leaves	Phenolic compounds	ChCl	Malic acid	1:1	30% (v/v)	0.1 g:1 mL	Magnetically stirred at 80℃	UAE at 40℃ for 30 min, 320 W	-The optimal RSM conditions were 40 °C for 15 min, 37% water, power at 144 W, and a solid:liquid ratio of 1:80 (w/v). -L-proline:glycerol showed the highest TPC.	Wu et al.^[Citation101]
			Glycerol
			Oxalic acid
			Xylitol
			Xylitol	1:2
			Levulinic acid
			Ethylene glycol
		Betaine	Levulinic acid	1:2
			Glycerol
		Citric acid	Glycerol	1:2
		L-Proline	Levulinic acid	1:2
			Glycerol	2:5
			Lactic acid	1:1
Soy products	Isoflavones	ChCl	D-glucose	2:1	30% (v/v)	1:1, 1:2, 1:3, 1:4, 1:5, 1:7.5 (mg/µL)	Stirred (600 rpm) at 70℃ for 15-30 min	UAE at 60 °C for 60 min, 440 W	The optimized RSM conditions were NADES based on ChCl:citric acid (1:1) at 60 °C for 60 min, 30% water, solid:liquid 1:3, and power 616 W.	Bajkacz and Adamek^[Citation102]
			L-tartaric acid	1:1
			Citric acid	1:1
				2:1
				1:2
			Saccharose	2:1
			Glycerine	1:1
				2:1
			D-xylose	2:1
		Urea	ChCl	1:1
			Citric acid	2:1
			L-tartaric acid	2:1
		Glycerine	D-glucose	2:1
			L-tartaric acid	2:1
			Citric acid	2:1
			ChCl: Citricacid: Glycerine	1:1:1
				2:2:1
Scutellaria baicalensis	Flavoniods	Citric acid	β-alanine	1:1	20, 30, 40, 50 and 60%(w/w)	100 mg:2 mL	Stirred at 50℃	Incubated at 40℃ for 1 h, ultrasonicated for 30 min at room temperature, and centrifuged for 20 min	The highest flavonoids yield was obtained with citric acid:β-alanine (1:1) with 50% water (w/w) and citric acid:proline (1:1) with 40% water.	Oomen et al.^[Citation103]
			Glucose
			Xylitol
			Proline
Rosemary (Rosmarinus officinalis L.)	Phenolic compounds	ChCl	Glycerol	1:2	10% (w/w)	150 mg:2.85 mL	Stirred at 50℃	UAE at 40±1℃ for 120 min, 50-60 khz	The highest TPC was obtained from ChCl:1,2-propanediol (62.21±3.85 mg GAE/g). ChCl:glycerol presented the highest value of DPPH.	Barbieri et al.^[Citation104]
			Oxalic acid	1:1
			Lactic acid	1:3
			1,2-propanediol	1:2
Wine lees	Anthocyanins	ChCl	citric acid		50-90% (w/w)	0.1 g:1 mL	Stirred at 80°C for 2-6 h	UAE at 35℃ for 15-45 min, 190-380 W, 37 khz	The optimized RSM conditions were an extraction time of 30.6 min, ultrasound power of 341.5 W, and 35.4% (w/w) water.	Bosiljkov et al.^[Citation105]
			oxalic acid
			malic acid	1:1
			glucose	2:1
			fructose	1.9:1
			xylose	2:1
			glycerol	1:2
Apple pomace	Phenolic compounds	ChCl	Lactic acid	1:1	50% (v/v)	2.5, 5 and 7.5% (w/v)	Stirred at 60℃	UAE at 60℃ for 2 h, 37 khz, 140 W	Sugar-based NADES (glucose:fructose:water) were the most effective NADES, followed by acidic NADES.	Deniz et al.^[Citation106]
				1:6
				1:9
			Malic acid	1:1			Stirred at 70℃
			Citric acid	1:1			Stirred at 80℃
				3:1
			ChCl: Sucrose:Water	1:1:11						Stirred at 50℃
			Glucose: Fructose: Water
			Fructose: Sucrose: Water
			Glucose: Sucrose: Water
		ChCl	Ethylene glycol	1:2			Stirred at 80℃
			Glycerol
			Urea
Apple pomace	Flavonoids and phenolic compounds	ChCl	Glycerol	1:2	30%	1:30 (w/v)	kept at 80℃ for 3 h in a shaking water bath	UAE at 40℃ for 40 min, 20 khz, 400 W	ChCl-based DES with glycerol, lactic acid, and citric acid performed better extraction than the conventional method. The highest TPC value was achieved using sonication at 40℃ for 40 min, 30% water, and a solid:liquid ratio of 1:30.	Rashid et al.^[Citation106]
			Lactic acid	1:3
			D-glucose	3:2
			Urea	1:2
			Citric acid	1:1
		Urea	Glycerol	1:2
		Tartaric acid	Glucose	1:1
Chinese wild rice (Zizania latifolia)	Flavonoids and phenolic compounds	ChCl	Glycerol	1:2	30% (w/w)	50 mg:2 mL	Magnetically stirred at 80℃	UAE at 50℃ for 20 min, 25 khz, 200 W	The optimal conditions were DES (choline chloride:1,4-butanediol) with UAE at a temperature of 51.2℃ and a solid:liquid ratio of 37.0 mg/mL.	Zeng et al.^[Citation107]
			1,4-butanediol	1:6
			D-fructose	1:1
			D-glucose	3:2
			Lactic acid	1:2
			DL-malic acid	1:1
			Urea	1:2
Helichrysum arenarium L.	Phenolic compounds	ChCl	Lactic acid	1:1	NADES:water, 3:1 (w/w)	500 mg:15 mL	Stirred at 80℃	UAE at 50℃ for 60 min (1st extraction) and 20 min (2nd extraction)	The optimized RSM conditions were an UAE time of 85 min, 38% water, and a molar ratio of ChCl:lactic acid of 1:4.	Ivanović et al.^[Citation108]
			Malic acid
			Malonic acid
			1,4-butanediol	1:6
			1,2-propanediol	1:2
			Glycerol
			Ethylene glycol
			ChCl: Fructose: Water	2:1:1
			ChCl: Maltose: Water	3:1:2
			ChCl: Glucose: Water	2:1:1
		ChCl	Urea	1:2
Kinkeliba (Combretum micranthum G. Don)	Phenolic compounds	ChCl	Lactic acid	1:2	20%	1 g:20 g DES	Stirred at 80℃ for 2 h	UAE at 25℃ from 30 min, 40 khz, 296W	ChCl:lactic acid showed the highest TPC values, followed by ChCl:acetic acid and ChCl:tartaric acid.	Zannou et al.^[Citation109]
			Tartaric acid					HAE: homogenized at 100 rpm, 25℃ for 30 min.
			Acetic acid					ME: at 25℃ for 12 h
Elderberry flower (Sambucus nigra)	Polyphenol	Glycine	L-lactic acid	1:5	DES:water 55, 70, 85% (w/v)	1 g:35 mL	Heated at 80℃ for 120 min	Pretreatment with ultrasound at ambient temperature (23 ± 1℃) for 5-40 min, 50 hz, 550 W. Extracted by stirring (500 rpm) at 50℃ for 150 min	The optimized RSM conditions were operated with stirring at 200 rpm, DES:water (85% w/v), and solid:liquid 1:60 (g:mL).	Kaltsa et al.^[Citation110]
				1:7
				1:9
				1:11
				1:13
Spent hops	Proteins	ChCl	Ethylene glycol	1:2	10 %wt	1:50 (w/w)	Stirred at 80℃ in an oil bath (20-40 min)	Extracted with trichloroacetic acid, acetone, phenol; centrifuged at 4℃	High protein yields were obtained from ChCl:ethylene glycol and ChCl:lactic acid, which varied between 40 and 64%.	Grudniewska and Pastyrczyk^[Citation111]
			Propylene glycol		5 %wt	1:40 (w/w)
			Glycerol			1:50 (w/w)
			Lactic acid			1:40 (w/w)
Microalgae (Chlorella pyrenoidosa)	Lipid	ChCl	Acetic acid	1:3	-	0.2:4.0 (w/w)	Stirred (≤800 rpm) at 80℃ for 2 h in water bath	Pre-treatment with DES at room temperature (28℃) for 5 and 10 min: manually shaken, stirred, and stirred with an ultrasonic homogenizer. Extracted with solvents (methanol and n-butanol)	The highest lipid yields (19.25%) were obtained using pretreatment with an ultrasonic homogenizer-assisted DES (ChCl:acetic acid, 1:3).	Ngatcha et al.^[Citation112]
Microalgae (Dunaliella salina)	Lipids and carotenoids	ChCl	Oxalic acid	1:1	*	20 mg:100 µL	Stirred at 80℃	Pre-treatment with DES: magnetically stirred (150 rpm) at ±29℃ for 24 h, followed by extraction with ethyl acetate and ethanol solvent	The total lipid recovery was 67.41%, achieved using a ChCl:urea-based DES without adding water to the system.	Asevedo et al.^[Citation113]
					20% (v/v)
			Urea	1:2	*
					20% (v/v)
			Glycerol	1:2	*
					20% (v/v)
Microalgae(Nannochloropsisgaditana)	Fatty acids(omega-3eicosapentaenoic acid)	ChCl	Lactic acid	1:2	-	0.5 g:45 g	Stirred at 80℃	Extracted at temperatures of 60 and 70℃ for 1.5 and 3 h	The fatty acid recovery percentage was higher (118%) with ChCl-ethylene glycol.	MorenoMartínezet al.^[Citation114]
			Ethylene glycol
			Glycerol
		Sodium acetate	Lactic acid	1:3
Dipsacus fullonum leaves	chlorogenic acid derivatives, flavone glycosides and iridoid glycosides	ChCl	Lactic acid	1:1-1:4	0-60%	-	Stirred at 80°C for 30-60 min	Shaken at room temperature for 1 h, then shaken in an ultrasonic bath at 40℃, 350 khz, 640 W for 30 min	-RSM and proposed optimized conditions: ChCl:lactic acid (1:2.4), 35% water, and a solid:liquid ratio of 1 g:27 mL.-The highest content of the extract was derived from flavone glycosides (0.47–2.28 mg/g), followed by chlorogenic acid (0.38–1.65 mg/g) and iridoid glycosides (0.17–0.92 mg/g).	Saar-Reismaa et al.^[Citation115]
		Choline acetate	Lactic acid	1:2	30%
		ChCl	Glycerol	1:1	30%
		Betaine	Lactic acid	1:2, 1:3	30%
		Menthol	Formic acid	1:1	*
			Acetic acid	1:1
			Lactic acid	1:1
			Formic acid, acetic acid, lactic acid	1:3 (1:1:1)
Sugarcane bagasse	Lignin	ChCl	Glycerol	1:2	-	1 g:10 mL	Stirred (300 rpm) at80-90℃	Heat at temperature range from 80-120℃ in sand bath for 2 h	The optimized conditions for lignin extraction were ChCl:glycerol at 135°C for 9 h of operation time and solid:liquid 1:23.41 (w/v), with obtained lignin yields of 70.31±1.91%.	Varilla-Mazaba et al.^[Citation116]
			Urea
Hemp bast	Cellulose fiber	ChCl	Urea	1:2	-	1:10 (w/v)	Heated at 80-90℃ for 1-2 h in an oil bath.	MAE at 100℃, 1000 kW for 5 min	The higher yield of cellulose (83.27%) was obtained from ChCl:urea.	Ahmed et al.^[Citation117]
			Glycerol
			Citric acid
		Sulfamic acid	Urea

*DES without water added, PEF-pulsed electric fields, MAE-microwave-assisted extraction, UAE-ultrasound-assisted extraction, HRE-heat reflux extraction, ME-maceration extraction, HAE-homogenate-assisted extraction.

Factors affecting extraction efficiency using DES

The physicochemical properties of DES are the main factors affecting the extraction efficiency of bioactive compounds. These properties include viscosity, polarity, density, solubility, and conductivity.^[118] Most DESs are high in viscosity as their intermolecular structures are held together by hydrogen-bonding networks. High solvent viscosity limits the interaction between the solvent itself and the target substances, decreasing mass transfer, thus extraction yield.^[66] Adding water to DES systems can significantly lower their viscosities.^[119]

Since the performance of a DES depends on its dissolving capacity, a high extraction efficiency can be assumed when the polarities of the solvent and the target compound are similar.^[120,121] Gao et al.^[118] demonstrated that ChCl-glycerol (1:2) was suitable for the extraction of phenolic compounds in mulberry leaves due to the similarity in their polarities. Addition of water into a DES system not only alters its viscosity as mentioned previously but also polarity. The presence of water could significantly affect extraction efficiency. Water could improve extraction yield by increasing concentration gradient between the solid and liquid phases.^[122] However, Huang et al.^[98] found that rutin extraction efficiency decreased when the NADES solution (ChCl: glycerol, 1:1) contained an excessive amount of water (>20%, w/w). The authors suggested that excess water disrupted the hydrogen bonds between the components of the DES, hence lowering the solubility of rutin in the DES system.

Molar ratio of HBA to HBD affects density, viscosity, and surface tension of the DES systems.^[123] The optimal HBA:HBD molar ratio can reduce the viscosity and surface tension, thus enhancing extraction yield. Gao et al.^[118] reported that improvement of phenolic compounds extraction from mulberry leaves was observed when the molar ratio of ChCl-glycerol DES was changed from 2:1 to 1:2. Lower yields were obtained at higher glycerol molar concentrations (from 1:2 to 1:5) due to decreasing interactions between target molecules and chloride anions.^[118]

To improve the extraction efficiency while maintaining the bioactivity of the target compounds, temperature and time must also be considered.^[32] An appropriate extraction temperature and time will produce optimal yields and retain bioactivity of the extractants.^[32,118] Optimal extracting temperature and time highly depend on the DESs used and the characteristics of the target substances. High temperatures allow penetration of solvent into the cells, promoting the interactions between DESs and target compounds. Viscosity of DES is temperature dependent. The higher the temperature the lower the viscosity. Lower viscosity of DES is observed at higher temperature due to the shift in interactions between anions and cations in the DES, leading to reorganization of the molecular structure.^[66] Although high temperatures encourage better contact between solvent and solute, the activity of thermolabile compounds is compromised.^[124] For example, temperatures above 50°C induce the decomposition and loss of PCs.^[125]

Purification of target compounds from DES

After extraction using DES, purification of the target compound from the solvent is a challenging step. Due to low vapor pressure property of DES, it is difficult to evaporate and separate the solute from the solvent.^[126] The strong intermolecular interactions between the target molecule and the DES network also present as a challenge for purification.^[127] Previous studies have reported several methods used to purify the extracted compounds from DES, such as back-extraction, anti-solvent extraction, solid-phase extraction, and adsorption chromatography with macroporous resins. Back-extraction has been applied to recover polymethoxylated flavonoids (PMFs) and hesperidin from a DES solution. A two-step back-extraction procedure with ethyl acetate as the first step agent and n-butanol as the second step agent successfully separated and recovered PMFs and hesperidin from DES solution. PMF and hesperidin recoveries were 95.87% and 86.32%, respectively.^[80] Huang et al.^[98] found that water was the most effective anti-solvent for recovering extracted rutin from NADES (95.1% recovery). Rutin in NADES was precipitated by adding water and keeping the solution at 0°C before centrifuging it. Another study on the recovery of the extracted ginsenosides from DES was achieved with the solid-phase extraction method (SPE). Ginsenosides in the glycerol: L-proline: sucrose (9:4:1) DES extracted phase were eluted with ethanol and evaporated, followed by LC-UV analysis for evaluation of the recovery efficiency (102.6 ± 4.1%).^[94] Separation of target substances by adsorption chromatography with different macroporous resins is also investigated. By using macroporous resin AB-8, the flavonoids from sea buckthorn leaves showed high recoveries from the DES solution (ChCl: 1,4-butanediol, 1:3) in the range of 72.36–84.99% and demonstrated strong antioxidant activity.^[128] The recovery of polyprenyl acetates from hydrophobic DESs (methyl trioctyl ammonium chloride: capryl alcohol: octylic acid, 1:2:3) was also accomplished using the macroporous resins AB-8 and DM130, which had efficient recovery yields of 76.52% and 74.08%, respectively.^[122]

Toxicity of DESs

While the precursors of DES systems are considered non- or low-toxic, the toxicity might still develop as a combining effect between the principal components. Studies on the toxicity and cytotoxicity of DES and NADES mixtures were carried out using in vivo animal models, including mice^[64,129] and Wistar rats.^[130] Study of DES toxicity on in vitro cell lines have also been published, such as on fish cells,^[131] human cells,^[64,132,133] human tumors,^[64,133,134] keratinocytes,^[135] and mouse cancer cells.^[133] Their toxicity against Gram positive and Gram negative bacteria, including Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella enteritidis,^[98] Bacillus subtilis and Pseudomonas aeruginosa,^[136,137] and Aspergillus niger strain^[138] were also reported. There are various factors considered to have impact on the cytotoxicity of DES, namely the combination of HBAs and HBDs, the molar ratios, the nature of DESs, the synergistic effects, and the interaction between solvents and cells.^[139] Chen et al.^[129] reported that ChCl-glycerol (1:2) was not toxic in mice, while Mbous et al.^[64] found that NADESs were more toxic than DESs due to their high viscosity and difficult circulation. NADES also affects Wistar rats; an excessive oral dose caused some deaths in samples.^[130] Bacterial and some cases of cell lines studies revealed that DESs were more toxic than each of their individual components.^{[137,140,141]} For human cells and mouse cancer cells, the preparation of DESs using organic acids as HBDs can increase toxicity while decreasing cell viability. However, more studies are needed to conclude if DES is non-toxic as the current viewpoint.

Applications of DES for extraction of cellulosic component, proteins, and polyphenolic compounds in BSG

Lignocellulosic component

Protic ILs exhibit selectivity in the solubilization of lignin, and concurrently maintain the integrity of cellulose structure.^[142] Ionic solvents destabilize the hemicellulose-lignin supramolecular hydrogen bonds, allowing the cell wall structure to be broken more easily.^[143] Having possessed such nature as an intrinsic property, DESs can be applied to separate lignin from cellulose and hemicellulose.^[144] Although DES are reported to be less effective than ILs in lignin solubilization, considerably high yield of high purity lignin still can be achieved.^[145]

The recovery of lignin from brewer’s spent grain using DES have been explored. Cassoni et al.^[146] used DES consisted of ChCl (HBA) and lactic acid (HBD) in a molar ratio of 1:5 for lignin extraction from 2 types of biomasses (BSG and olive tree pruning (OTP)). With the optimum conditions for extraction (120°C and 5 h), BSG yielded a higher lignin content (54.4%) compared to that from OTP (37.8%). Both samples are rich in lignin and showed high purities of the extracts (>75%). However, BSG lignin exhibited a significantly lower antioxidant activity than OTP lignin. The influence of HBD and extraction temperature on lignin extraction with high β-O-4 content from brewer’s spent grains was studied.^[147] Choline chloride-based systems with two different types of HBD (lactic acid, LA) and glycerol (Gly) were investigated at moderate extraction temperatures (60 and 80°C). Pretreatment with the acidic DES (ChCl: LA) showed higher lignin yields compared with ChCl: Gly pretreatment, demonstrating that the acidity of DES has an impact on the yield of lignin. The yield also increased at higher temperature (34.5% at 60°C and 39.3% at 80°C). This could be due to the effect of temperature in lowing DES viscosity, enhancing mass transfers and improving the DES performances.^[147] However, the β-O-4 bonds in lignin can be affected by temperature.^[148] The NMR spectra indicated that the content of β-O-4 decreased when the pretreatment temperature increased.^[149]

Proteins

DES has been used in both solid-liquid and liquid-liquid protein extraction. Applications of DES on recovery of protein from plant sources have been done extensively, while those from animal sources are quite limited.^[74] Hydrogen bond donor has a significant effect on the yield and properties/functionality of the extracted proteins.^[150,151] Low polarity DES are found to be more effective in protein precipitation and facilitate the extraction of hydrophobic amino acids.^[151,152] In terms of applying DES to extract BSG protein, at present there was only on report by Wahlström et al.^[153] who investigated 6 potassium and sodium carboxylate salt – urea DESs systems. High protein yields (almost 80%) were obtained from DES systems of sodium acetate: urea (1:2) and potassium acetate: urea (1:3), with 10% water addition (by weight). The extraction was achieved at 80°C for 4 h. The addition of water could be the reason for the lower melting point and melting enthalpy of DES compared to the mixture without water. The protein yield obtained by DES extraction (79%) was almost double to that obtained by the traditional alkaline extraction process (41%) previously reported.^[154]

Polyphenolic compounds

The most common solvents used for recovering phenolic compounds, from natural sources, are methanol and ethanol. Recently, the extraction of phenolic compounds using ChCl- based DESs has been studied.^[155] The HBA and HBD composed in DES or NADES mixtures act as both Bronsted-Lewis acids and bases.^[156] According to Dai et al.,^[157] the interactions of hydrogen bonds between ChCl-DES and phenolic compounds might be related to the extractability of phenolic compounds. It may also involve mass transfer from the solid to liquid phase and the dissolution of phenolic compounds in DES due to their polarity.^[155] High-polar polyphenols is firstly leached from solid particles, followed by less-polar substances.^[158] The presence of hydrogen bonding between DESs and phenolic compounds prevents oxidative degradation by lowering the movement of phenolic molecules, resulting in decreased contact time with air.^[159] Focusing on recovering phenolic compounds from BSG, the screening of four different DESs in order to find the most effective one was studied by López-Linares et al.^[160] Choline chloride-based DES was prepared with glycol, lactic acid, glycerol, or 1,2-propanediol in a molar ratio of 1:2. The most effective DES, choline chloride with glycerol, was carried out with microwave assistance (700W, at 65°C for 20 min). Optimization of the extraction parameters was then investigated on the selected DES using central composite design. The parameters studied included temperature (50–100°C), time (5–25 min), and percentage of water in the DES systems (20–70%). The total phenolic content of 2.89 mg gallic acid/g BSG was obtained using choline chloride: glycerol under 100°C for 13.30 min and 37.46% (v/v) of water in the DES mixture. The yield obtained was higher than using the conventional solvent (80% methanol) (1.2 mg gallic acid/g BSG). Ferulic and coumaric acids were the most abundant phenolics identified. The details of recent research on the applications of DES for bioactive compound extraction from BSG are presented in Table 5.

Table 5. Applications of DES or NADES for extraction of BSG.

Target compound	DES/NADES			Water content	Solid:Liquid	Preparation method	Extraction method	Result	Reference
	HBA	HBD	Molar ratio
Lignin	ChCl	Lactic acid	1:5	5, 10, 15, 20, and 50% (v/v)	1:10 (w/w)	Stirred (100 rpm) at 60°C for 10–15 min	Extracted at temperatures 60, 90 and 120°C for time 1, 3 and 5 h	Lignin yield was 54.4% with purity of 77.2%,and TPC was 88.8 ± 9.2 mg GAE/g lignin.	Cassoni et al.^[Citation146]
Lignin	ChCl	Lactic acid	1:10	-	1:20 (w/v)	Stirred at 80°C for 2 h	Magnetically stirred at 60 or 80°C for 24 h in an oil bath	Lignin extraction yields of 39.3% and 34.5% were obtained from DES ChCl: lactic acid at80 and 60°C, respectively.	Provost et al.^[Citation147]
		Glycerol	1:2
Protein	Sodium acetate	Urea	1:2	with and without 10 wt% of water	-	Stirred at 80°C	Extracted at 80°C (90°C for sodium formate:urea mixtures) for 1, 2, 4 and 20 h.	High extraction yields of 79% and 74% were obtained with sodium acetate: urea in molar ratios of 1:2 and 1:3, respectively, and also with potassium acetate: urea (1:3) (78%).	Wahlström et al.^[Citation153]
			1:3
	Potassium acetate		1:2
			1:3
	Sodium formate		1:2
			1:3
Phenolic compounds	ChCl	Ethylene glycol	1:2	20–70% (v/v)	10% (w/v)	Stirred (500 rpm) at 80°C for 30–90 min	MAE at 65°C for 20 min, maximum power 700 W	The optimum condition of microwave-assisted DES extraction was observed with ChCl:glycerol at 100°C for 13.30 min and 37.46% (v/v) water in the DES, which contained TPC of 2.89 mg gallic acid/g BSG.	López-Linares et al.^[Citation160]
		Lactic acid
		Glycerol
		1,2-propanediol

Conclusions

Previous studies have shown an increased interest in using DES alone or in combination with emerging green extraction technologies to extract bioactive compounds from various sources. However, using DES for BSG extraction has not been studied extensively, particularly for lipids. Therefore, the applications of DES to the recovery of bioactive compounds from BSG are needed in future studies in more detail in order to expand its applicability. For upscaling, DES extraction should be studied and developed to convert batch extraction to continuous extraction and make it more suitable for large-scale or industrial applications.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by EU through H2020 under Grant 952594; and Nujamee Ngasakul thanks Czech University of Life Sciences Prague for PhD scholarship.