Deep Eutectic Solvents and Natural Deep Eutectic Solvents for Extraction and Purification of Proteins from Animal and Botanical Sources - A Review

The rising global population has led to an escalating demand for affordable, high-quality proteins intended for human consumption. Different efforts have been made to develop efficient and greener solvents for protein production. Most proteins are produced from raw plant and animal parts, but the use of the residues in commercial-scale protein production is very scarce. Therefore, this research aims to collect an overview of deep eutectic solvents (DESs) and natural deep eutectic solvents (NADESs) for protein extraction and purification. In this context, solvent type and operating parameters should also be selected to modify DESs and NADESs physicochemical characteristics for the achievement of high protein yield with preserved functional properties. The results show that appropriate implementation of combined DESs and NADESs with advanced extraction and purification techniques can improve protein yield and prevent detrimental effects on the extracted protein and environment. The application of DESs and NADESs can increase the efficiency of protein extraction and recovery in various parts of plants and animals by 55.72% and 98.16%, respectively, with purity reaching 99.82%. This research also reviews safety, environmental impacts, and drawbacks as well as shows feasible future recommendations for commercial-scale protein production processes. NADESs are safer than petroleum-based solvents but have higher toxicity than DESs. Magnetic adsorbent and magnetic solid-phase extraction methods have been shown to reduce labor-intensive steps, resulting in shorter operating times and superior protein recovery while maintaining functional properties. Meanwhile, protein producers' knowledge and motivation to use DES and NADES are strengthened.

Animal and plant parts; Deep eutectic solvent; Extraction; Operating parameter; Protein

    Protein is the basic unit of living organisms acquired from sustainable origins, namely animals, plants, and microorganisms. Since the world’s population is forecasted to reach 9 billion by 2050, the demand for affordable high-quality protein is expected to increase (Grudniewska et al., 2018). Therefore, effcient, greener and sustainable methods for protein production receives a growing interest. The underused sources have also attracted attention for the preparation of affordable pure proteins. In this context, the food industries may also adopt underused plant and animal residues by extracting and refining proteins as valuable biomacromolecules showing numerous health benefits to reduce environmental problems and support sustainable development goals.

The crucial step in using protein is to adopt suitable extraction technologies to acquire protein from animal or plant matrices. Conventional procedures are techniques regularly used, such as chemical methods. However, the methods can lead to reduced extraction yields due to protein degradation (Kumar et al., 2021b). Numerous industries also continue to depend on traditional methods because of financial viability. Chemical methods are categorized according to petroleum-based solvents, water, alkalis, organic solvents, and acids (Bowen et al., 2022; Kumar et al., 2021b). Meanwhile, chemical methods are used to enhance the retrieval of proteins (Kumar et al., 2021a; Bose et al., 2019). Protein purification methods include precipitation through the addition of an agent, electrophoresis, ion exchange, and affnity chromatography (Lin et al., 2021). Conventional acid-based extraction is less promising due to lower quality of protein (Kumar et al., 2021b). Petroleum-based and solvent-based chemical methods have been used in the food and pharmaceutical industries for centuries. However, this solvent has serious problems, those related to low extract yield and coextraction of unwanted substances, long extraction time, operational complexity, expensive, toxicity, biodegradability, fammability, safety and environmental sustainability, denaturation or reduced biological activities (Ling and Hadinoto, 2022; Fuad, Nadzir, and Harun, 2021; de Jesus and Filho, 2020).

Organic solvents are crucial in protein extraction and precipitation processes, leading to the production of pure products. Water-based extraction is a widely used method for obtaining proteins from different sources (Chen et al., 2019a). The process is commonly adopted because of the elevated solubility and stability of isolated protein, which is mostly attributed to high content. Protein with the capacity of attaching to lipids have non-polar or polar side chains and contain aromatic amino acids that readily dissolve in organic solvents such as ethanol, butanol, and acetone (Qiaoyun et al., 2017). Aqueous two-phase systems, created by combining polyethylene glycol (PEG) with salts can be used efficiently for purification and separation of proteins (Asenjo and Andrews, 2011).

Alkalis such as NaOH and KOH are frequently used to maintain a basic pH and enhance the yield compared to organic extraction. Altering the pH to a basic level caused the disulphide linkages to dissolve, leading to an enhancement in recovery and yield (Contreras et al., 2019). The solubility is enhanced by an elevation in the pH of solvent due to the ionization of acidic and neutral amino acids at high pH levels. Therefore, extracting protein in an alkaline environment results in greater protein yields. In this context, a yield above 90% can be obtained from soybeans, rapeseed, and other seed commodities through alkaline extraction at high pH. Alkaline enhances protein extraction and disrupts the structure of amino acids, lysine and cysteine. This significantly degrades the digestibility and acceptability of the extracted protein (Kumar et al., 2021b).

To address this limitation, the food industries have initiated the use of greener solvents as substitutes for petroleum-based solvents, alkalis, organic solvents, and acids to manufacture products (Ling and Hadinoto, 2022; Kumar et al., 2021a; Kumoro et al., 2019). In addition, ionic liquids (ILs), deep eutectic solvents (DESs) and analogues from natural sources (sugars, acids, and amino acids) known as natural deep eutectic solvents (NADESs) are progressing as advanced agents to decrease the adverse impacts of petroleum-based solvents (Ling and Hadinoto, 2022). DESs are composed of a quaternary ammonium salt (QAS) and a hydrogen bond donor (HBD), such as a carboxylic acid or alcohol. These components are mixed in specific molar ratios to form a eutectic mixture with a lower melting point (Abbott et al., 2003). Meanwhile, NADESs consist of natural compounds such as sugar (glucose), organic acids (citric acid), and amino acids (choline). This compound forms a eutectic mixture with properties similar to DES but derived from renewable resources (Dai et al., 2013b).

Despite having comparable physical characteristics to ILs, such as high viscosity, low volatility, chemical and thermal stability, as well as non-flammability, DESs are not made of ionic compounds. Non-ionic chemicals can produce DESs (Ling and Hadinoto, 2022), which are more cost-effective with higher biodegradability (Kudlak, Owczarek, and Namiesnik, 2015). These solvents are environmentally friendly, easy to synthesize, cheap, biodegradable, and possess low toxicity (Chen et al., 2018).

The use of DESs and NADESs as an environmentally friendly solvent for extracting valuable compounds has gained significant interest in recent years. This is due to the distinct characteristics, including customizable properties, versatility, ease of preparation, unique super-molecular structure that shows strong attraction to different compounds, high solubility, and stabilizing capability (Zannou and Koca, 2022; Dai et al., 2013a). The majority of investigations on extraction have mostly focused on bioactive small compounds (Pratiwi et al., 2020; de Faria et al., 2017). Several reviews have been published on the latest developments in extraction of bioactive small compounds using DESs. Bonacci et al. (2020) gave an example showing the ability to extract DESs from tiny molecules such as phenolic compounds. The use of choline chloride glycerol DESs resulted in a significantly higher recovery yield of oleuropein (~88,287 ppm) from olive oil processing wastes. This was achieved in 10 minutes, which is twice the usual water extraction method requiring 30 minutes. The research reported by de Faria et al. (2017) also observed similar performance of DES. In this context, higher recovery of polyphenols and flavonoid compounds was obtained from saffron processing wastes compared to conventional solvents such as aqueous methanol, aqueous ethanol, and water. Mulia et al. (2015) have also succeeded in extracting mangostin with the highest yield of 2.6% (w/w) from dried mangosteen skin using NADESs. This was obtained using a mixture of choline chloride and 1,2-propanediol with a mole ratio of 1:3.

Even though DESs and NADESs extraction of small bioactive compounds is highly practical, the investigation of biological macromolecules, such as proteins, oils and carbohydrates, has gained attention (Ling and Hadinoto, 2022; Mulia et al., 2018). Considering the distinct physicochemical characteristics of proteins in comparison to other small or large molecules, the function of DES and NADESs should be reported in extracting proteins derived from animals and plants. The selection of solvents depends on factors such as the solubility of protein, extraction efficiency, and desired properties of solvents. There are differences in protein from animals and plants due to variations in biochemical composition, cellular structure, and properties. Animal protein extraction includes methods such as aqueous, organic solvent, or enzymatic digestion, depending on the tissue type and desired protein (Malva et al., 2018). Meanwhile, plant protein extraction requires additional steps to break down cell walls, such as mechanical disruption or enzymatic treatment, followed by aqueous or buffer solutions (Wang, Liu, and Lu,  2013). Extraction from animals and plants with greener and environmentally friendly solvents is interesting for further analysis.

In this research, extraction of DES and NADESs from animals and plants is presented. The review commenced with a discussion of the fundamental and physicochemical characteristics of DESs and NADESs suitable for protein extraction processes. The main results and trends observed in solvent-based protein extraction were discussed with the safety and environmental aspects.

Ionic liquids (ILs) are used as solvents for reaction or extraction processes and provide numerous advantageous characteristics, such as high boiling point, broad liquid range, selective dissolving capacity, excellent thermal stability, non-flammable and molecular structure variety (Morais et al., 2020). However, the preparation is very complicated, requiring high equipment, manufacturing costs, and laborious recovery from the mixture with the target analyte (Ling and Hadinoto, 2022). To overcome the limitation, DESs are developed as a new generation of advanced greener solvents.

As a eutectic mixture, DES can be synthesized partially or completely from non-ionic compounds, which function as hydrogen bond donors (HBDs) such as betaine, choline chloride, guanidine hydrochloride and proline, as well as hydrogen bond acceptors (HBA) including amine groups (ethanolamine, dimethylamine, and imidazole), polyols (ethylene glycol, propylene glycol, and glycerol), and carboxylic acids (acetic, citric, maleic, and lactic acids) (Saini et al., 2022). Meanwhile, NADESs consist of natural compounds such as sugar (glucose), organic acids (citric acid), or amino acids (choline). This compound forms a eutectic mixture with properties similar to DESs but derived from renewable resources (Dai et al., 2013b). The preparation comprises sequential procedures, such as size reduction, heating, evaporation, and low-temperature drying (Saini et al., 2022). After careful selection of the prerequisite substances, homogenization is carried out at ideal conditions (i.e., <100°C) to attain a eutectic mixture and equilibrate the resulting solution with ambient conditions (Mišan et al., 2020). Therefore, the physicochemical characteristics of DESs are equivalent to ILs as a perfect substitute. The macromolecular structures are appropriate for protein extraction concerning solubility, affnity, and stability (Landa-Castro et al., 2020). The influence of the composition of HBAs and HBDs on the characteristics is explained in Section 4.

DESs and NADESs can exist as binary or ternary mixtures. In addition, NADESs possess simpler preparation procedures, better efficiency, selectivity, biodegradability, thermal stability, and sustainability (Ijardar, Singh, and Gardas, 2022). Based on chemical compositions and formulas, DESs are grouped into four classes (Abbott et al., 2004).

3. DESs and NADESs Physicochemical Characteristics Related to Extraction Process

Even though the physical characteristics of DESs are closely similar to the conventional ILs, the chemical characteristics are significantly different. The influential physicochemical characteristics are melting point, freezing temperature, polarity, solubility, miscibility, pH, density, viscosity, interfacial tension, refraction index, and ionic conductivity (Omar and  Sadeghi, 2022b). The anion size, equilibrium molar ratio of HBA/HBD, alkyl chain length, and molecular mass of HBA/HBD at the melting point determine the physicochemical characteristics (Omar and Sadeghi, 2022a). However, viscosity and polarity possess significant effects on extraction performance (Tolmachev et al., 2022). Interfacial tension also has a remarkable effect on liquid-liquid and ultrasound-assisted extraction processes (Kumoro et al., 2022).

The fluidity of DESs is mainly characterized by viscosity (Omar and Sadeghi, 2022b), influencing the solubility of target samples. In this context, the biomacromolecules are more soluble in less-viscous DESs. Temperature, type of HBAs and HBDs, HBA/HBD molar ratio, and molecular mass greatly affect viscosity. Meanwhile, an increase in temperature appreciably decreases viscosity due to hydrogen bond network cleavage between HBA and HBD as well as the reduction of internal resistance of molecules (Ling and Hadinoto, 2022). DESs possess higher viscosity when sugar, carboxylic acid and metallic compounds are selected as the HBD, while less viscous type can be prepared using ethylene glycol, glycerol, and phenol (Ling and Hadinoto, 2022). After water addition, the viscosity decreases, facilitating better mixing and mass transfer leading to a higher extraction performance. An extreme amount of water ceases the advantageous characteristics of DES. Therefore, an appropriate water quantity should be selected to prevent the disruption of hydrogen bond interactions among DES-forming substances (Ling et al., 2020).

Polarity, which is closely related to Hansen’s solubility parameter (HSP) is a theoretical method used as an introductory tool to estimate the solubility of target biomacromolecules in different solvents and select the most appropriate DESs and NADESs for extraction from respective natural sources (El-Kantar et al., 2019). Since DESs are mostly polar substances, the superior ability to dissolve the target analytes can improve extraction yield (Gullón et al., 2020). Eutectic solvent polarity is attributed to the HBDs used for the synthesis and the highest are usually synthesized from carboxylic acids. In contrast, polyols and sugars result in eutectic solvents with the lowest polarity (Xu et al., 2019a). The dilution of DESs with water beyond 50% causes more hydrogen bond destruction and leads to a decrease in extraction ability (Dai et al., 2015).

The density of DESs is higher than pure water (Omar and Sadeghi, 2022b), with the value ranging from 1.0 to 1.35 g/cm³ at 25^oC. However, the density of metallic salts containing DESs lies between 1.3 to 1.6 g/cm³ (Zhang et al., 2012), depending on the molecular arrangement, temperature, HBA/HBD molar ratio, as well as the presence of cavities and voids. Temperature greatly decreases the density, refraction index, and acoustic velocity due to the enhancement of ionic movement and unoccupied volume (Omar and Sadeghi, 2022b).

Surface tension is an important characteristic of DESs utilization in the interface and colloid system (Kumoro et al., 2022). In this context, HBDs and HBAs have an appreciable influence on the surface tension of DESs. Since DESs are highly hydrophobic, the surface tension is enhanced greatly when the water mole fraction is higher than 0.9 (Chen et al., 2019b). However, the surface tension decreases as a function of solvent concentration when petroleum-based solvents or crystal water in the salt component are added with an increasing temperature from 20 to 60°C (Chen et al., 2019b). 

The majority of DESs are highly viscous liquids with large ion sizes and possess low ionic conductivity (below 1 mS/cm at ambient temperature) which limits the application in isoelectric-based protein precipitation (Omar and Sadeghi, 2022b). The ionic conductivity is influenced by alkyl chain length of cation, the characteristics of organic salt, HBDs and HBAs ionic ratio, temperature, and water content (Dai et al., 2015). Therefore, the conductivity can be improved by increasing the free volume through the reduction of cation size and the substitution of HBD with fluorinated substances to reduce viscosity (Abbott, Capper, and Gray, 2006). Another strategy is to increase the temperature which helps to break the hydrogen bond network and increase ionic motility (Omar and Sadeghi, 2022b). The tunable physical characteristics, specifically density, viscosity, ionic conductivity, and freezing point obtained by a careful selection of biodegradable HBD and HBA couplings make DESs and NADESs more attractive for specific applications (Zhang et al., 2012).

4.    DESs and NADESs as Extracting Media for Protein Extraction and Purification

DESs, formed by the combination of hydrogen bond donors and acceptors, have been extensively studied for the versatile applications. For instance, Abbott et al. (2003) showed the exceptional solvent properties of DESs, particularly in extraction of bioactive compounds from natural sources. The results showed that DESs, such as those composed of choline chloride and urea, reported superior extraction yields and selectivity compared to conventional solvents. Similarly, Dai et al. (2013a) suggested the efficiency in catalyzing various chemical reactions, showing the potential as green reaction media. Conversely, NADESs are derived from naturally occurring compounds such as sugars, organic acids, and amino acids, and the efficiency has been reported in various processes. For example, Socas-Rodríguez et al. (2021) investigated the use as extraction solvents for bioactive compounds from different matrices. The research showed that NADESs offered a greener alternative to conventional solvents, with comparable or improved extraction efficiency. Generally, DESs and NADESs offer promising prospects as efficient and greener solvents across various industries. The unique properties, coupled with the renewable and sustainable nature, position solvents as viable alternatives, contributing to the development of more sustainable processes.

Efficiency and yield when using DES or NADES can vary depending on the specific application and the characteristics of solvent system. These eco-friendly solvents offer several advantages contributing to improved efficiency and yield in various processes, including extraction, synthesis, and catalysis. According to Smith, Abbott, and Ryder (2014), solvents possess unique properties in enhancing the efficiency and yield of chemical processes. The properties include the ability to enhance solubility, provide selective extraction, operate under mild reaction conditions, and offer recyclability. By leveraging the characteristics, DES and NADES have shown potential to facilitate higher yields and improved efficiency compared to conventional solvents (Smith, Abbott, and Ryder, 2014).

To elaborate on a specific example, extraction of bioactive compounds is considered from plant material using NADESs. Choi et al. (2011) reported the use in understanding cellular metabolism and physiology, emphasizing the potential for efficient extraction of target compounds from natural sources. The selected method includes preparing the NADESs by mixing components such as choline chloride and organic acids in specific ratios, followed by extraction of bioactive compounds from plant material at an appropriate temperature. The resulting extract can be analyzed using analytical techniques to quantify yield and purity. Moreover, Dai et al. (2015) discussed the tailoring of properties, including the addition of water, to optimize performance in various applications. This optimization process enhances efficiency and yield by fine-tuning solvent characteristics to suit specific extraction or synthesis requirements.

In the case of protein extractions, the most used DESs belong to type III and comprise ChCl as HBA and amines, amide, carboxylic acids, sugars, and polyols as HBD (Zhang et al., 2012). Ling and Hadinoto (2022) showed that there were considerable variations in melting points when the HBA:HBD ratios in ChCl:urea were changed. A 1:2 ratio resulted in a significantly lower melting temperature of 12^oC, while a 1:1 ratio generated NADESs with a high melting point of 50^oC. Therefore, the choice of HBD component has a major impact on the melting point of the resulting DES as well as the effect of the molar ratio of HBA and HBD. For example, the use of ethylene glycol, citric acid, malonic acid, oxalic acid, xylitol, and glycerol as HBD produced DESs with melting temperatures ranging from 69 °C to room temperature. In this context, the type of HBD affects the melting point of the synthesized DESs (Zhang et al., 2012). An appropriate HBA/HBD molar ratio in the mixture plays a crucial role in ensuring the suitability of the resulting DESs to selectively dissolve the targeted proteins and easy solvent recovery.

4.1. DESs and NADESs for Solid–Liquid Extraction of Protein from Plant and Animal Parts

The mechanism of DESs and NADESs in solid-liquid extraction of proteins from plant and animal parts includes a series of steps. These solvents penetrate the cell structures of plant materials or animal tissues, disrupting hydrogen bonds and hydrophobic interactions (Morais et al., 2020). Within plant or animal material, DESs and NADESs solubilize proteins through interactions such as hydrogen bonding and electrostatic interactions (Bowen et al., 2022). Meanwhile, extraction methods such as shaking or stirring enhance protein leaching from the solid matrix into solvent phase (Zhou, Fakayode and Li, 2023). Subsequent phase separation techniques, such as centrifugation or filtration, isolate protein-containing solvent phase from the solid debris (García et al., 2015). Protein recovery from solvent phase is also achieved through precipitation or purification methods, with the possibility of solvent regeneration for further use (Abbott et al., 2004). This mechanism shows the potential of DESs and NADESs as sustainable alternatives for protein extraction, offering applications in various industries including food, pharmaceuticals, and biotechnology.

Different investigations have also focused on the usage of biomass residues as tabulated in Table 1. These greener solvents have proven the ability to achieve higher extraction performance in terms of product yield and purity. Liu et al. (2017) prepared DESs by mixing several HBAs, such as ChCl, glycine, betaine, alanine chloride, acetylcholine chloride, and nicotinic acid with PEG200 to extract pumpkin seeds protein. The PEG200-based DESs were mixed with a four-fold volume of ethanol as well as 1 M hydrochloric acid to control the pH at 4.5 and recover 93.8% of the extracted protein (Table 1). Currently, extraction of proteins using PEG-based DESs is growing more rapidly. This is because the extracted proteins are more stable and accepted by Food and Drug Administration (FDA), which opens wider opportunities for more sustainable developments in the agricultural, food and pharmaceutical sectors (Morgenstern et al., 2017). Even though PEG-based DESs show excellent affnity to protein as well as a remarkable capacity to ease precipitation, more research is required to ensure the scale-up and practical application at a commercial scale.

Hernández-Corroto et al. (2020) used highly polar and hydrophilic DESs derived from ChCl and acetic acid to extract pomegranate peel protein. The resulting extract contained 19.2 mg protein/g with high antihypertensive activity, which was stronger than petroleum-based extract. After selecting nine ChCl – diol mixtures as DESs, Yue et al. (2021) found that ChCl–1,4-butanediol/water mixture was the perfect solvent for oat protein extraction with an effciency of 55.72% as well as better stability and foaming capacity (Table 1). DESs derived from multicomponent mixtures could precipitate protein more rapidly than binary mixtures caused by high partitioning capacity and polarity.

Chen et al. (2021) prepared DESs by mixing ChCl with glycerol to extract soy protein with a yield of 10% higher than the acid-based precipitation method, which reported enhanced heat resistance and hydrophobicity. An equivalent observation was stated by Lin et al. (2021) when acidic DESs based on ChCl and levulinic acid were used to extract protein from bamboo shoots. The research observed a more profound protein yield enhancement (60%) compared to extraction using sodium hydroxide solution.

With the intention of marine by-product valorization, Rodrigues et al. (2021) prepared DES by blending betaine and propylene glycol in a ratio of 1:3 to recover proteins from sardine fish heads and entrails. A yield of 162.2 mg protein/g fish parts greater than the common aqueous extraction was obtained. This product contained numerous hydrophobic amino acids, namely alanine, isoleucine, leucine, and valine that are applicable to produce less polar DESs. In addition, the resulting extracts showed stronger antioxidant and antimicrobial capacities. The existence of hydrophobic DESs components promotes more intensive interactions between the respective proteins with the cell membrane of the assayed microorganisms.

Rodrigues et al. (2021) extracted protein from sardine processing residue using betaine–propylene glycol and obtained a yield of 162.2 mg protein/g fish with higher antioxidant and antimicrobial activities than the water extract. DESs made from lactic acid and L-cysteine are capable of recovering keratin from coarse wool residue without altering the polypeptide structure (Okoro et al., 2022). Based on those reviewed facts, DESs and NADESs are capable of extracting valuable proteins and preserving desirable functional characteristics.

Table 1 Extraction of proteins from sustainable sources using various DESs and NADESs

There are many parameters, which provide essential roles during biomacromolecules extraction, specifically proteins using DESs and NADESs, such as temperature, duration, solvent-to-solid ratio, solid particle size, and pH (Kumoro et al., 2022). An appropriate selection of temperature, water content, and pH will alter polarity, solubility, interfacial tension, and viscosity supporting the achievement of high extraction performance (Huang et al., 2017).

4.2. DESs for Liquid-Liquid Extraction of Protein from Plant and Animal Parts

Aqueous two-phase systems (ATPS) are used to facilitate selective liquid-liquid extraction of proteins by homogenizing a water-soluble polymer with another inorganic salt, such as PEG-salt-water mixture and ethylene oxide–propylene oxide or copolymer–polyoxyethylene detergent at a concentration higher than the critical value (Xu et al., 2016). Xu et al. (2016) prepared an ATPS by mixing ChCl and glycerol with a salt solution and successfully extracted 98.16% of bovine serum albumin (BSA) from the DESs phase without any protein conformation changes. Hydrogen bonding, salting out, and hydrophobic interactions are possible characteristics facilitating protein uptake (Xu et al., 2016).

Xu et al. (2019b) recovered lysozyme (Lyz) from chicken egg white using DESs-based ATPS derived from tetrabutylammonium bromide (TBAB), glycolic acid (Gly) and Na₂SO₄ salt. More than 98% of the lysozyme was transferred to the DESs-phase with 91.73% of the initial activity preserved. Similarly, Meng et al. (2019) selected a mixture of tetrabutylammonium chloride (TBAC), L-proline – xylitol (Pro– Xyl) of 1:6 and polypropylene glycol 400 as DESs and ATPS to extract chymotrypsin from the mixture with BSA and lysozyme. The result also showed that 97.30% chymotrypsin was accumulated in the [Pro][Xyl]-rich phase under optimum conditions (pH 7.0, 35^oC, 12 minutes shaking). The phase separation capacity of DESs can be improved by enhancing the alkyl side chain length of carboxylic acids and the addition of a benzyl group.

Liquid-liquid extraction of proteins using DESs and NADESs in combination with ATPS includes a sophisticated mechanism. Solvents penetrate the cellular structures of plant materials or animal tissues, disrupting intermolecular forces and solubilizing proteins (Abbott et al., 2003). Subsequently, the mixture is combined with an aqueous solution to form a biphasic system, where protein partitions between the two phases based on the physicochemical properties (Singh and Tavana, 2018). Liquid-liquid extraction methods such as stirring or shaking facilitate the transfer of protein into solvent-rich phase, while the remaining sample matrix remains in the aqueous phase. Subsequent separation methods, such as centrifugation or inversion isolate protein-enriched phase from the aqueous phase (Mendes et al., 2023). Protein recovery can be achieved through precipitation or purification methods, with the potential for solvent regeneration for reuse. This integrated method obtains the advantages of DESs and NADESs in combination with ATPS to offer an efficient and sustainable method for protein extraction from diverse biological sources.

4.3. Protein Purifcation Using DESs and NADESs

The extracted proteins must be purified to allow food, pharmaceutical, and nutraceutical industries to commercialize safe products for actual human and animal consumption. The well-established methods to purify protein are alkali, ammonium sulphate or acetone precipitation, salting-out, ion exchange, electrophoresis, and affnity chromatography (Sindhu et al., 2012). However, the established techniques suffer from limitations, such as protein activity loss, denaturation or complexation, higher operating costs, and equipment operation difficulty. For example, an enzyme's activity can be distorted by excessive interaction with a polar solvent. In this context, a smart purifcation strategy using ATPS can be a potential alternative method due to shorter purification and phase separation time, excellent capacity to preserve biological activity, great biocompatibility, low toxicity, and lower requirement of water (Gai et al., 2011). DESs have also been used as a part of ATPS to purify protein (Table 2) (Zeng et al., 2016).

Purification process using ATPS offers a versatile method for separating biomolecules based on differential partitioning between two immiscible aqueous phases (Albertsson, 1970). This strategy includes the design of phase-forming components such as polyethylene glycol (PEG) and dextran to optimize protein partitioning behavior (Singh and Tavana, 2018). After mixing the sample with ATPS, the target protein preferentially partitions into a phase while impurities remain in the other, facilitating efficient separation (Hatti-Kaul, 2000). Smart technologies integrated into purification process enable real-time monitoring of protein, allowing for precise adjustment of conditions to enhance separation efficiency (Bernau et al., 2022). Meanwhile, separation techniques such as centrifugation are used to isolate the phases containing the purified protein, followed by further purification steps when necessary (Du et al., 2022). By obtaining automation and data-driven optimization, this ATPS-based strategy ensures high yields and purity levels in biotechnology and pharmaceutical applications.

Table 2 Utilization of DES-based ATPS for protein purification

To achieve the objective, Li et al. (2016) adopted betaine as the HBA to compose six types of DESs using urea, methyl urea, glucose, sorbitol, glycerol, and ethylene glycol as HBD to extract and purify BSA from protein mixture. In this context, betaine – urea mixture was found as the most favorable DESs in combination with ATPS for BSA extraction and purification from the complex systems where an effciency of 99.82% was achieved. Moreover, Xu et al. (2015) used DESs derived from ChCl as HBA and ethylene glycol, glycerol, glucose, and sorbitol as HBD to extract and purify BSA from the mixture. The result confirmed that ChCl–glycerol mixture at a 1:1 molar ratio was the most favorable DESs. Under optimum pH, temperature, and time, BSA and trypsin recoveries were 98.71% and 94.36%, respectively. Meanwhile, Xu, Wang, and Hou (2020) observed that ChCl–urea (1:2) mixture was the most preferred DESs to extract R-phycoerythrin from red algae. A ternary mixture of ChCl–urea–K₂HPO₄ was the suitable ATPS to purify R-phycoerythrin with a separation effciency of 92.60% (Xu, Wang, and Hou, 2020).

5.    Limitations of DESs and NADESs as Proteins Extraction and Purification Media

Even though many chemicals comply with the functions of HBD and HBA to form DESs, some of solvents are not appropriate for protein extraction from natural sources. Therefore, a careful selection should be carried out to determine the appropriate DESs, specifically those related to recovery and isolation from the phase (Smith, Abbott, and Ryder, 2014). DESs viscosities and densities decline with the increase in temperature but the ionic conductivity rises (Lores et al., 2017). These opposite features can be the critical problems of viability and adaptability potentially affecting the utilizations by the industry.

Efficient recovery and isolation are essential for protein separation from DESs to enable recycling. Xu et al. (2015) observed that recovery from this solvent was slow since the process occurred under a mass transfer regime due to high interfacial resistance. Even though salt concentration is modified by mixing DESs with a freshly ethanolic saline solution mixture, only 32.9% of protein is recovered. Therefore, more advanced improvements should be addressed to the existing protein back extraction and DESs recovery techniques to attract more interest from enterprises (Li et al., 2016).

6.    Safety and Environmental Aspects Associated with The Utilization of DESs and NADESs

NADESs are less toxic than petroleum-based solvents, and the adverse effects on human health remain unclear but solvents are regarded as safe (GRAS). A previous in vivo research on mice and Wistar rats confirmed that NADESs reported higher toxicity than DESs, which was rooted in high viscosity values. At high concentrations, solvent becomes very viscous limiting the circulation and inducing acute effects, liver failure, and mortality (Benlebna et al., 2018). Ecotoxicity research of DESs reported disparate sensitivities to the tested ecosystem models, which mainly varied with the composition of DESs. The results showed that DESs and NADESs toxicity was affected by some parameters, namely viscosity, HBD/HBA molar ratio, organic acids content, pH, type of cells or organisms, and synergistic effects.

Previous safety analysis is required before using DESs and NADESs for food and medicinal applications. Since the characteristics largely depend on the combinations of the components, a compendious database of toxicological properties of DESs should be set up which supports the numerous component variations of the mixtures with computational predictive approximations. In addition, most NADESs combinations and applications are patented limiting the industrial applications.

7.    New Methods for Solid-Phase Extraction Using DESs and NADESs

The direct utilization of DESs and NADESs using traditional extraction methods can collaborate with ultrasound-assisted (UAE) and microwave-assisted extraction (MAE) to achieve higher protein yield and purity. The application can be expected to extensively break cell wall structure and ease the release of intracellular protein from plant and animal matrices (Chemat et al., 2019). Correspondingly, DESs can also be developed in MAE for the recovery of proteins and show greater performances than conventional techniques (Bubalo et al., 2016). This pilot strategy possesses various advantages mainly related to higher efficiency, shorter operation time, and lower solvent requirement than conventional extraction methods using petroleum-based solvents.

To enhance extraction performance, magnetic adsorbents can be incorporated into ATPS, which function to adsorb proteins for pure protein recovery (Liu et al., 2012). In magnetic solid-phase extraction (MSPE), nanoparticles are dispersed into extraction medium to adsorb proteins. The analytes are immediately segregated from the magnetic adsorbents with the aid of an external magnetic eld enabling nearly complete recovery of protein molecules and recycling of nano adsorbing particles (Table 3) (Wen et al., 2016). This innovative method eliminates some lengthy and laborious stages, namely centrifugation and filtration, that result in a shorter operating time, and exceptional protein recovery with preserved functional properties and offers an important function in purification procedures (Huang et al., 2015).

Table 3 Application of magnetic particle-modified DESs for protein purification

    In conclusion, DESs and NADESs were reported to show higher extraction efficiency, improved bioactivity, better recyclability, reusability and biodegradability potential, as well as less toxicity than conventional organic solvents. The collaboration of solvents with green chemistry-based industrial processes to reclaim valuable substances from plant and animal sources reported more favourable results compared to the available conventional solvent extraction processes. In addition, some important process parameters, namely solid and solvent ratio, temperature, mixing, pH, and duration, significantly affected the effectiveness of DESs during extraction processes. From a scale-up and industrial application perspective, the use of DESs required further research concerning thermal stability, analyte purification, solvent recovery, operational cost, toxicity, and environmental impacts.

    The authors acknowledge Universitas Diponegoro for its financial assistance under the third-year term of World Class Research Universitas Diponegoro (Kategori A) with contract No.: 118-17/UN7.6.1/PP/2021.

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