Application of Enzyme-Assisted Extraction for the Recovery of Natural Bioactive Compounds for Nutraceutical and Pharmaceutical Applications

Abstract

Enzyme-assisted extraction (EAE) involves the use of hydrolytic enzymes for the degradation of the cell wall or other cell components. This supports the diffusion of the solvent into the plant or fungal material, leading to easier elution of its metabolites. This technique has been gaining increasing attention, as it is considered an eco-friendly and cost-effective improvement on classical or modern extraction methods. Its promising application in improving the recovery of different classes of bioactive metabolites (e.g., polyphenols, carotenoids, polysaccharides, proteins, components of essential oil, and terpenes) has been reported by many scientific papers. This review summarises information on the theoretical aspects of EAE (e.g., the components of the cell walls and the types of enzymes used) and the most recent discoveries in the effective involvement of enzyme-assisted extraction of natural products (plants, mushrooms, and animals) for nutraceutical and pharmaceutical applications.

1. Introduction

For thousands of years people have used natural products for food, prophylactics, cosmetic purposes, and to treat diseases. Almost half of drugs originate from or are inspired by natural molecules. Natural medicines and ethnocosmetics are common in primary health care systems and are everyday choices in developing countries. On the other hand, growing interest in phytotherapy and natural products can be observed in developed countries. Therefore, in addition to the therapeutic, care, and pro-health facets, acquisition of natural metabolites has an economic aspect [1,2,3]. This is related to their widespread use in the food, cosmetic, and pharmaceutical industries. In each industry, great importance is attached to the effective extraction of active metabolites from natural material. Moreover, the trend of using environmentally friendly technologies is becoming stronger [4].

Extraction is designed to separate bioactive compounds from raw materials. Primarily, different variants of solvent extraction are used. Chemicals diffuse into the solvent, and the extract is collected. The greater the diffusivity and solubility of the active substances, the greater the extraction efficiency is. The improvement of extraction parameters is influenced by many factors, not only the chemical nature, size of particles, the type and amount of the solvent but also the temperature and the type and time of extraction. Classic extraction methods are often less effective; they require a relatively long extraction time and a larger amount of solvent. These are, e.g., maceration, reflux extraction, and percolation [5]. New extraction methods, e.g., ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), micellar extraction (MAME), or supercritical fluid extraction (SFE), are often characterized by higher speed, reduced solvent consumption, and higher elution of bioactive compounds [6].

Regardless of the extraction method used, there are some natural mechanical barriers hindering the extraction process. These include cell structures that need to be overcome to elute the metabolites of interest. Many components of the cell wall, such as lignins, celluloses, or some proteins, give cells strength but constitute an impediment during extraction of bioactive components. Some active metabolites are present in the cytoplasm, some are stored in vacuoles or plastids, and others are bound in a polysaccharide–lignin network by hydrogen bonds, which makes them not always available for conventional solvents [7,8,9]. In every case, the enzyme-assisted extraction method allows reducing the resistance of natural matter. The EAE is based on the use of enzymes that catalyse the cleavage of covalent bonds in the presence of water. This disintegrates cell structures and increases the permeability of the material. The enzyme-assisted extraction can be a standalone method or a pretreatment for conventional extractions. Enzymes are highly specific in appropriate conditions. Particle size, time, pH, and temperature must be considered. Enzymatic reactions proceed effectively at a relatively low temperature and moderate pH and in a relatively short time (up to few hours) and do not require expensive equipment. The duration and mild conditions allow minimizing degradation or isomerisation of active molecules. In the majority of cases, a positive or even highly positive effect of enzymatic pretreatment on extraction efficacy is observed [10,11,12,13]. Optimization of key conditions often results in lowering the costs and energy consumption of the whole extraction process [14,15]. Moreover, enzymolysis takes place in a water solution or in a buffer [16]. Therefore, no additional portion of organic solvent is needed. All the abovementioned conditions make the enzymatic pretreatment of the solution match the assumptions of green chemistry. In some cases, additional positive effects can be noticed, e.g., co-extraction or more effective extraction of value-added phytochemicals (fatty acids and phenolics during distillation of essential oil) [13]. Summarizing all the above aspects, enzyme-assisted extraction shows many economic benefits with a high efficiency, which will be highlighted below [17].

2. The Principles and Enzymes Used in EAE

As mentioned above, the majority of utilized phytochemicals or other bioactive natural compounds are hidden inside the cell and/or are linked with their macromolecules. Thus, cell walls and membranes constitute a specific barrier that must be overcome.

2.1. Components of Plant and Fungal Cell Wall

A plant cell wall is a complex structure serving various functions in the plant cell. Among other things, it is a protective barrier and supports cell division and differentiation. The cell wall is mechanically strong; hence, it can maintain the internal turgor pressure. It shows flexibility in response to stress stimuli and activates genes responsible for enzymes that change the structure of the cell [18].

During plant growth, the primary cell wall, which provides structural integrity by balancing high tensile strength with flexibility needed for cell growth, is formed [18]. Cell walls allow plant cells to achieve large increases in volume and a variety of shapes. When a cell has finished growing, it can build a secondary cell wall to specialize in specific functions depending on the type of cell [19]. The secondary cell wall is stronger and thicker. This structure is located between the cell membrane and the primary cell wall [18]. The secondary cell wall strengthens plant tissues and structures, ensuring impermeability and mechanical reinforcement [19].

Depending on the plant species, cell walls differ in their chemical components and structures. This is related to the evolution and differentiation of their composition to adapt plants to various terrestrial habitats [18].

The major components (>90% dry weight) of the cell wall are polysaccharides. The main biopolymer is cellulose. This insoluble carbohydrate is present in both primary and secondary cell walls. Cellulose is responsible for the wall’s mechanical strength [20]. Cellulose-rich cell walls are a hallmark of vascular terrestrial plants. This polysaccharide is called a carrier polymer due to the nanostructure it forms in the cell wall. It consists predominantly of unbranched chains of multiple D-glucose subunits linked by β-(1,4) bonds [18]. Glucan chains arrange in a specific way, creating an amphiphilic structure. The outward facing hydroxyl groups form two polar surfaces, and the exposed sugar skeleton forms two apolar surfaces [19]. Cellulose chains containing up to 25,000 glucose residues have a high degree of polymerization. These chains combine with hydrogen bonds to form regular assemblies, thus creating ordered microfibrils.

The second most common polysaccharide fraction consists of hemicelluloses. These non-cellulosic polysaccharides cross-link cellulosic microfibers, thereby preventing microfibrils from collapsing and sliding over each other [21]. Hemicelluloses are heteropolysaccharides with a low degree of polymerization. They form short branched chains. Various monosaccharides can be present in the hemicellulose structure. Typical components for the hemicellulose chain are glucose, mannose, and xylose linked by β-(1→4) bonds. They usually combine with other components of the cell wall through ionic and hydrophobic interactions as well as covalent or hydrogen bonds [22].

Pectins are branched heteropolysaccharides found in the cell wall. They contain acidic (e.g., galacturonic acid and glucuronic acid) and neutral monosaccharides (e.g., rhamnose, galactose, and arabinose). Pectins are mainly found in primary plant cells and in laminae. They provide rigidity and integrity to plant tissues. Pectins participate in intercellular adhesion and maintain the water level in the walls of plant cells. They take part in the defence mechanisms of the cell wall against pathogens. Galacturonic acid is the most common monosaccharide in pectin chains. The most abundant pectin structural polymer is homogalacturonan. This chain consists of galacturonic acid units linked linearly by an α-1,4 bond. Other common pectin polysaccharides are rhamnogalacturonan I and ramnogalacturonan II [21]. The skeleton of rhamnogalacturonan I is composed of repeatable disaccharide units, which include galacturonic acid combined with rhamnose. Rhamnogalacturonan I shows structural flexibility in different types of cells and at different levels of development. It is common for L-arabinan and D-galactan to attach to rhamnosyl residues. In contrast, galacturonic acid residues are intensively acetylated. Rhamnogalacturonan II is the most complex pectin polysaccharide. In its skeleton, there are four oligosaccharide side chains (A–D) and over 20 bonds with 12 different sugars. The A side chain is an octasaccharide that can be methylated. The B side chain can consist of six to nine sugars with different terminal residues and different acetylation degrees. In turn, disaccharides are the C and D side chains [18].

Another important component of cell walls is lignin. It is a non-polysaccharide fraction. Lignin is an amorphous polymer consisting of monomers derived from phenylalanine. This composition is characteristic of vascular plants. In grass, monomers are derived from tyrosine [18]. This complex phenyl biopolymer is found in secondary cell walls in association with cellulose and hemicellulose in such specialized tissues as the cortex, vessels, and fibres. Lignin stops cell growth and is responsible for the mechanical strength of the plant and protection against abiotic stresses as well as pathogens and herbivorous animals [21].

The structure of the cell wall of other natural sources of bioactive molecules, e.g., fungi, is different. In the case of the fungal cell wall, it is based on fibrous carrier polysaccharides that constitute scaffolding for the matrix elements. The structure of the cell wall ensures plasticity and, at the same time, mechanical strength. Fungal hyphae use the force generated by turgor pressure to penetrate the substrate [23].

Fungal cell walls show great variability in their composition and organization. They serve a number of important functions in helping fungi to grow, morphogenise, and survive. Thanks to cell wall sensor proteins, the fungus evaluates and reacts to changes in the environment. In this way, the cell wall provides protection against a range of unfavourable environmental conditions, such as drying, cold, heat, or osmotic stress. It is a protective barrier against microorganisms. The cell wall of pathogenic fungi is responsible both for protection against the defensive systems of host cells and for the adhesive properties needed for invasion. It thus plays a key role in virulence and pathogenicity. The adhesive properties are also crucial in the colonization of new environments by fungi. Various polysaccharide polymers, proteins, and carbohydrate–protein complexes may exist in fungal cell walls to form a cross-linked matrix. The main components are chitin, melanin, glycoproteins, β-1,3-glucan, and β-1,6-glucan [23,24,25]. However, there are species-specific differences in quantitative, qualitative, and structural aspects [23].

Chitin is a structural polysaccharide composed of linear β-(1,4) N-acetylglucosamine chains [23]. Chitin chains can be hydrogen bonded to form extremely tensile microfibrils, in which chitin molecules are arranged antiparallel to each other. Chitin is largely responsible for the rigidity and integrity of the cell wall but is not an essential ingredient in all types of fungal cells. In some fungi, some chitin is deacetylated and chitosan is formed. This polymer of glucosamine residues has better solubility than chitin [24].

Melanins are high molecular weight pigments consisting of polymerized phenolic and/or indole compounds [23]. These amorphous polymers are located in the cell wall and protect against damage associated with ultraviolet radiation, because they intensively absorb UV light. Melanin, similarly to lignin, provides stiffness, hardening of the cell wall, and protection. Therefore, it can be found in structures exposed to adverse conditions, e.g., fungal ascopores or conidia. Melanin is an important virulence factor; it ensures the strength of conidia during penetration and increases the resistance of fungal cells to lysis in host tissues [24].

Besides the abovementioned most common polymers, there is a large group of species-specific ones. Moreover, other components, e.g., proteins or polysaccharide–peptide complexes, can build a physical barrier hindering elution of natural bioactive compounds or are connected to molecules of interest [23,25]. Hence, effective hydrolysis of their bonds can be employed during the EAE process. Such an approach is found in the extraction of plant, fungal, or animal products [6,26,27].

2.2. Types and Activity of Hydrolytic Enzymes

Due to the multilayer structure of cell walls, there is a possibility of using several groups of enzymes, which act on different polymers, to decompose the wall and reach the metabolites contained in the cell. Cellulases, pectinases, hemicellulases, and proteases are the most commonly used enzymes. They are abundantly produced by microorganisms and plants; therefore, they can be manufactured and purchased for scientific or industrial use.

2.2.1. Cellulases

Cellulase is not a single enzyme; in fact, it is a complex of enzymes, which act synergistically. The complex comprises at least three major subclasses of enzymes such as endoglucanases (endo-1,4-β-_D-glucanase), exoglucanases (cellobiohydrolases; 1,4-β-_D-glucan cellobiohydrolase), and β-glucosidases (cellobiase, β-_D-glucoside-glucohydrolase) [28,29]. Endoglucanases have an ability to randomly hydrolyse β-1,4-glycosidic bonds inside the molecule and, consequently, release a long chain of cello-oligosaccharides. These enzymes act on the cellulose amorphous region. Exoglucanases break cello-oligosaccharides at the end of the chain and release glucose and cellobiose. Cellobiase catalyses the conversion of cellobiose into glucose [29,30].

Cellulases are used in a number of industrial processes, e.g., wastewater treatment, the bakery, textile, brewing, paper, and food industries, wine production, and the production of bioethanol. These enzymes are naturally produced abundantly by microorganisms and cultures of higher fungi, e.g., Trichoderma reesei, Aspergillus niger, Trichoderma viride, Penicillium purpurogenum, Penicillium echinulatum, Pleurotus xorida, Pleurotus cornucopiae, and Bacillus pumilus [31,32,33]. Interestingly, the activity of an enzyme at specific temperatures and pH values depends on the organism that produced this enzyme. For example, cellulase obtained from Penicillium purpurogenum, Pleurotus xorida, and Pleurotus cornucopiae exhibited maximum activity at a temperature of 50 °C. The optimum pH value for fungal cellulase is 4.5. However, cellulase from Aspergillus phoenicus has its maximum activity at 4.8–5.5 pH [28].

Trichoderma reesei, also known as Hypocrea jecorina, is considered the most efficient producer of cellulase for industrial applications [28]. The enzyme is produced through submerged fermentation of a selected strain of the fungus and can be bought as an aqueous solution for laboratory research. Cellulase obtained from T. reesei is able to decompose cellulose into glucose, cellobiose, and other glucose polymers as well. According to the producer, its specific activity is estimated at more than 700 units/g, and its density is 1.10–1.30 g/mL. The optimum values of pH and temperature are estimated to be 6 and 52 °C, respectively (www.sigmaaldrich.com; access on 18 January 2022).

The other cellulase that is quite popular is cellulase from Aspergillus niger. The enzyme is available in the form of powder, and its specific activity is estimated at more than 0.3 units/mg. Cellulase from Aspergillus niger hydrolyses endo-1,4-β-_D-glycosidic linkages in several types of polysaccharides. The stable pH range of the enzyme is 5.0–10.0 and the optimal pH for its activity is 6.0–7.0. It has been established that the enzyme obtained from A. niger cultivated in a liquid culture medium with rice bran and yeast extract is thermally stable, and the optimal temperature at pH 6.0 is 70 °C [34]. However, the investigations conducted in 2020 by Sulyman et al. revealed that cellulase obtained from A. niger cultured on Arachis hypogeae shells had an optimum pH and temperature of 4 and 40 °C, respectively [35]. This may indicate that the activity of specific enzymes depends on the producing species and culture conditions. Aspergillus sp. cellulase obtained through submerged fermentation can also be bought as an aqueous solution, and its specific activity reaches over 1000 units/g.

The growing interest in the potential and efficient applications of cellulases influences the direction of scientific research. Many new studies are focused on improving the production of cellulase by different organisms, e.g., bacteria [29,36].

2.2.2. Pectinases

Pectinases are the most widespread enzymes found in fungi, bacteria, and plants [37]. They are responsible for the degradation of pectic substances. Their potential use in, e.g., the food industry, production of juices and wines, and many others has been indicated as well [15,37]. Interestingly, the use of pectinases strengthens the taste and the colour of wine [37]. Moreover, these enzymes are used to reinforce and accelerate tea fermentation and to remove the mucilaginous layer of coffee beans [30]. Similarly to cellulases, pectinases are not a single molecule but a mixture. Pectinases comprise protopectinases, pectin methyl esterases, pectin acetyl esterases, polymethylgalacturonases, polygalacturonases, pectate lyases, pectin lyases, rhamnogalacturonan rhamnohydrolases, rhamnogalcturonan galacturonohydrolases rhamnogalacturonan hydrolases, rhamnogalacturonan lyases, rhamnogalacturonan acetylesterases, and xylogalacturonan hydrolase [37]. Pectinases can be produced by a number of microorganisms, such as Aspergillus sp., Streptomyces sp., Penicillium chrysogenum, and Bacillus sp. [37,38,39]. For instance, acid pectinase commonly used in extraction and clarification is produced by the fungi Aspergillus niger [37]. As mentioned previously, enzymes from different organisms exhibit different activity in various conditions. For example, fungal polygalacturonases have high activity at pH 3.5–5.5 and an optimal temperature between 30 and 55 °C, whereas polygalacturonases from Bacillus licheniformis and Fusarium oxysporum have an optimum pH of 11. The optimum pH for pectin lyases is estimated at 4.0–5.0. Pectin lyases from Aspergillus sp. are efficient at pH 5.5 and a temperature range of 40–50 °C [30,40,41]. In turn, pectate lyases need higher pH values (between 7.5 and 10) for their optimal activity. Similarly, enzymes from Ewinia sp. achieve their activity at pH 6, whereas this enzyme from B. licheniformis is active at pH 11 and requires an optimal temperature in the range of 40–50 °C [30]. It is worth noting that the enzyme from B. licheniformis needs calcium ions for its activity.

Pectinases can be purchased in liquid or powder form and their efficiency is product-dependent. Pectinase from Aspergillus aculeatus is in a liquid form with declared activity of approx. ≥3.800 units/mL. However, the enzyme from Aspergillus niger shows specific activity of >1 U/mg and is commercially available as a powder. Pectinase from Rhizopus sp. can also be purchased as a powder with specific activity estimated at 400–800 units/g solid (www.sigmaaldrich.com; accessed on 18 January 2022).

2.2.3. Hemicellulases

Hemicellulases is a common name for several enzymes with hemicellulolytic activity, e.g., xylanases, glucuronidases, arabinofuranosidases, galactosidases, mannanases, and acetyl or feruloyl esterases. These classes of hemicellulases can hydrolyse different structures of hemicellulose [30].

The properties of hemicellulases depend on their origin. Interestingly, different organisms are able to produce different sets of enzymes, which may act synergistically [30,42]. Xylan is a structure that occurs in the largest amount in hemicellulose; hence, endoxylanases and xylosidases are the most important enzymes. Endoxylanases (endo-β-1,4-xylanase/1,4-β-_D-xylan xylanohydrolase) decompose glycosidic bonds releasing small oligosaccharides. Xylosidases (1,4-β-_D-xylan xylohydrolase) hydrolyse β-1,4-bonds, which leads to the release of xylose from xylooligosaccharides. Another enzymatic compound from the hemicellulase family is β-mannanase (endo-1,4-β-mannanase/1,4-β-_D-mannan mannanohydrolase), which decomposes hemicelluloses with a dominant number of mannans. Due to this action, it releases short mannooligomers [30]. Subsequently, mannooligomers can be hydrolysed by another enzyme—β-mannosidase (β-_D-mannosidase/1,4-β-_D-mannoside mannohydrolase), which leads to the release of mannose. α-L-arabinofuranosidase, in turn, belongs to debranching enzymes, which “cut off” side groups or substituents [30]. α-_D-glucuronidases cleave α-1,2- bonds between the main chain in glucuronoxylan and glucuronic acid residues, which may lead to debranching of glucuronoxylans [43]. Another enzyme, i.e., acetyl xylan esterase, is an important agent used for enzymatic hydrolysis of lignocellulosic materials, since it removes O-acetyl groups from acetylxylan, supporting or enhancing xylanase activity [44]. Among hemicellulases, there is α-1-_D-galactoside galactohydrolase, which hydrolyses α-1,6 bonds from the main chain and releases alpha-1,6-linked-_D-galactopyranosyl substituents. The list of hemicellulase enzymes also comprises phenolic acid esterases: feruloyl esterases and p-coumaryl esterases, which hydrolyse ester bonds between arabinose and monomers of ferulic acid as well as bonds between arabinose and p-coumaric acid [30].

Depending on the origin of the enzyme, there may be different optimal pH and temperature values. As reported by Thomas et al., fungal xylanase is more effective at pH values ranging from 3.5 to 6.5 and temperatures of 40–60 °C [45]. In turn, bacterial xylanases are more effective at pH 5.0–8.0 and in the temperature range of 50–80 °C [46].

Hemicellulases are produced abundantly by microorganisms, but fungi and thermophilic bacteria, e.g., Aspergillus sp., Trichoderma sp., Bacillus sp., and Cellvibrio sp., are the most essential for commercial use [42]. Interestingly, Clostridium bacteria are able to produce a complex of cellulases and hemicellulases. The enzymes are also produced by Penicillium and Rhizopus sp. These enzymes are used in the bakery and beverage industries. They can replace oxidative substances and can also be used to clarify juices. They are also used in the textile industry and for production of prebiotic oligosaccharides [30,42]. Aspergillus niger hemicellulase is a commercially available product. It is in a powder form and its specific activity is estimated at 0.3–3.0 unit/mg solid. According to the manufacturer, one unit will produce a relative fluidity change of 1 per 5 min using locust bean gum as a substrate at pH 4.5 and 40 °C. Importantly, the enzyme should be stored at a temperature of −20 °C (www.sigmaaldrich.com; accessed on 18 January 2022).

2.2.4. Proteolytic Enzymes

Proteolytic enzymes known as proteases or peptidases catalyse the hydrolysis of amide bonds in peptide substrates. However, the biological and catalytic functions of different proteolytic enzymes are diverse. They are present in, e.g., Lactococcus genera, Clostridium histolyticum, Porphyromonas gingivalis, and Bacillus licheniformis bacteria [47]. In bacteria, they play a key role in supplying cells with nitrogen compounds necessary for their growth [48]. Proteases are also produced by mammals, viruses, bacteria, fungi, protists, animals, and some plants [49].

Proteolytic enzymes can be used for many biotechnological purposes and in many branches of industry. As the group of proteolytic enzymes is not homogenous, there are several ways to classify them. They can be grouped according to the source of production, details of the catalysed reaction, mechanism of action, homology, and molecular structure. The large group of proteolytic enzymes comprises aspartic/aspartyl peptidase, cysteine peptidase, serine peptidase, metallopeptidase, threonine peptidase, glutamic peptidase, and asparagine peptide lyase. Aspartic peptidase shows optimum activity at pH 3–6 and can be produced by Endothia parasitica and species of Penicillium, Aspergillus, Rhizopus, Mucor, and Rhizomucor. The enzyme for industrial application is produced by R. miehei. Another type of peptidase is a cysteine peptidase also known as thiol peptidase. Its activity requires a cysteine residue in the active site. Due to the presence of a thiol group and its tendency to be oxidized, the enzyme needs a reducing agent for its action. The maximal pH value for the activity is 4.5–7 (www.americanchemicalsuppliers.com; accessed on 21 January 2022).

2.2.5. Commercial Mixtures of Enzymes

The application of synergistically acting enzymes is often crucial for their efficient utilization due to the complexity and heterogeneity of cell structures. Another situation is the need to increase the effectiveness of enzymatic extraction. Therefore, attempts to use different combinations of enzymes have been undertaken. For example, it has been found that the combined use of papain, pectinase, and cellulase in a ratio of 1:1:1 in the extraction of polysaccharides from the fruiting body of Tricholoma matsutake is very effective [50]. Hence, there are several commercial enzymatic mixtures available (e.g., Viscozyme L^®, Lallzyme^®, Kemzyme^® Plus, Multizyme^®, and Ultrazym^®). Viscozyme L^® is a multienzyme complex containing arabinase, cellulase, β-glucanase, hemicellulase, and xylanase. According to the Viscozyme L^® producer, it is an effective enzyme for extraction of polyphenols from unripe apple. Its activity was estimated at ≥100 FBGU/g. It can be bought as a liquid and should be stored at a temperature of 2–8 °C (https://www.sigmaaldrich.com, accessed on 18 January 2022; www.kemin.com, accessed on 18 January 2022; https://biosolutions.novozymes.com; accessed on 19 January 2022).

Another enzyme mixture available on the market is Lallzyme^®, which contains pectinase, endocellulase, and galactanase (https://www.lallemandwine.com; accessed on 1 February 2022). The product can be used in wine production.

Another product is Kemzyme^® Plus, which contains xylanase, β-glucanase, and cellulase. These are just a few examples of available enzyme mixtures that can be helpful in increasing the efficiency of extraction or enhance the economic viability of production in food industry (https://www.kemin.com; accessed on 18 January 2022).

3. Studies on Enzyme-Assisted Extraction of Metabolites from Natural Materials

Enzymolysis has been recently investigated as an improvement of classical (e.g., maceration and distillation) or modern (supercritical fluid extraction, ultrasound-assisted, microwave-assisted, or surfactant-assisted extraction) extraction methods [11,13,51,52]. Interestingly, its application has been examined in a variety of natural products, i.e., algae, higher plants, fungi, and animals [6,50,53,54,55,56]. EAE has been used to develop new solutions for management of natural materials (e.g., fruits) and post-production wastes, e.g., soy pulp or waste sesame bran [10,52,57]. It is considered a promising tool for obtaining larger portions of products or active ingredients for the food, cosmetic, and pharmaceutical industries and related fields. Table 1 shows examples of studies of the enzyme-assisted extraction of different types of natural materials and their ingredients. The optimized conditions are specified as well.

The EAE technique has been applied for extraction of low molecular compounds (e.g., phenols and polyphenols, oils and fatty acids, essential oil, sugars, di- and triterpenes, vitamins) and macromolecules (polysaccharides, proteins) [14,52,58,59,60]. Many plant and fungal species belonging to a variety of plant and fungal families were pre-treated with enzymes to obtain larger portions of their metabolites. Different hydrolytic enzymes and enzymatic mixtures (including commercial enzyme preparations) were investigated, including cellulases, hemicellulases, pectinases, and proteases [6,51,58,61,62]. The majority of the research evaluated the total amount of eluted compounds belonging to a particular group of metabolites (e.g., total amount of polyphenols, flavonoids, lipids, carbohydrates, proteins, etc.) [11,13,58,63]. A smaller number of studies concerned the extraction of individual compounds.

Enzymolysis was relatively often employed to enhance extraction of plant phenols and polyphenols [11,63]. In almost all cases, it contributed to increased release of total phenolics or target compounds, in comparison with untreated samples (up to several tens of percent) [11,63,64]. Cellulase, pectinases, and different types of enzymatic mixtures (e.g., Viscozyme and Kemzyme) were revealed to be the most effective [11,65].

Hydrolytic pretreatment with proteases, pectinases, cellulases, and β-glucanase was shown to result in the higher efficiency of extraction of lipids and lipophilic compounds from plants and algae [59,66]. It resulted in high oil recovery with a high concentration of lipid-soluble vitamins [59]. However, in some cases of EAE, it was less effective than organic solvent extraction [67].

It has been shown that EAE with proteolytic and cellulolytic enzymes can exert a significant effect on the extraction of proteins, polysaccharides, and reducing sugars from plants, animals, and fungi. The application of this technique did not adversely affect the biological activity of samples [68,69].

The enzymolysis step allows extraction of comparatively higher levels of essential oil from the plant material. It has been shown that the yield of essential oil can be improved even 30–40 times in the case of Forsythia suspensa or Coriandrum sativum fruit [13,60]. A similar effect was observed when EAE was implemented for extraction of triterpenes from liquorice root or Pseuderanthemum palatiferum leaves [14,62]. It was also a promising tool for elution of stevioside from stevia leaves [70].

A high extraction yield was achieved in all studies of the use of EAE in elution of carotenoids. In the case of carrot pomace powder, the extraction efficiency was improved up to 90%. This effect was obtained with commercial pectinase (Endozym Pectofruit) [15]. Another enzymatic mixture was shown to be effective in improving the elution of lycopene from tomato industrial waste [71]. Pectinase contributed to a high yield of astaxanthin from freshwater chlorophyta [53]. All these carotenoids are obtained and used in large quantities in the food, cosmetic, and pharmaceutical industries.

Similarly, other industrially relevant molecules from beetroot (betalains, betacyanin, and betaxanthin) or ginger rhizome (oleoresin and 6-gingerol) can be more effectively obtained with the use of enzymatic pretreatment [16,72].

It has been observed that changes in the extraction process affect the yield, chemical composition (amount and proportions of metabolites, elution of by-products), and biological activity of the obtained extract/product [13,16,62,73].

Efficient enzymatic hydrolysis requires specified conditions related both to the enzyme and to the natural product (enzyme type, enzymatic mixture; temperature, pH, time, enzyme concentration, sample particle size, and water–to–material ratio). In the case of more lipophilic metabolites, the enzymatic pretreatment is often only the first step, followed by a proper extraction procedure involving the use of organic solvent/-s or equipment [13,61,71]. Therefore, the majority of studies of novel applications of enzyme-assisted extraction suggest the need for optimization of conditions and parameters. Employment of statistical methods (response surface methodology (RSM) and orthogonal test design) is a frequently chosen solution, often with great success [14,56,74]. Cellulolytic enzymes can be inhibited or even inactivated by several factors, e.g., products of hydrolysis (cellobiose and glucose), oxidants, reductants, phenolic compounds, some solvents and ions (Hg²⁺ and Cu²⁺), or surfactants [28,34,75,76]. This should be taken into account when designing or optimizing the enzymatic procedure.

Most papers have shown a positive effect of hydrolytic pretreatment on the efficiency and usefulness of the whole process. However, there also reports where enzyme-assisted extraction was less effective that other methods [73,77,78]. These “negative” effects were explained by the difficulties faced by the enzyme, e.g., enzyme–substrate interactions, release of the plant proteases reducing enzyme activity, or by lack of optimization of, e.g., the water–to–material ratio or the enzyme concentration [14]. In many cases, mixtures of enzymes were found to be more effective than a single enzyme.

Table 1. Examples of studies on enzyme-assisted extraction of metabolites from natural materials (plants, mushrooms, and animals).

Natural Material	Target 1	Optimized Conditions of Enzymolysis and Extraction	Extraction Effects and Yields in Optimal Conditions	Ref.
Polyphenols and phenols
Annona squamosa L. (Annonaceae) fruits	Yield and quality of juice; total phenolic content	Pectinase Enzyme concentration 2.21% Temp. 47 °C Time 4.47 h pH 4.9–4.36	Yield 88% (w/w) Total polyphenols 93.95 ± 0.51 μg of GAE/mL	[58]
Coriandrum sativum L. (Apiaceae) residue from the seed distillation process	Total phenolic (TPC) and total flavonoid (TFC) contents antioxidant activity	Enzymolysis: Cellulase (10 mg/100 g) Time 1 h Temp. 40 °C Hydrodistillation in Clevenger-type apparatus Time 2 h	Increased release of phenolic compounds improved antioxidant activity	[13]
Crocus sativus L. (Iridaceae) tepals	Total phenolic content and total anthocyanin content	Enzymolysis: Mixture of cellulolytic and hemicellulolytic preparations (1:1) Enzyme concentration: 0.12–0.15% Solvent–to–sample ratio 10:1 (v/w) Time 145–185 min Stage 1—rehydration: Time 1 h pH 4.0 Temp. 25 °C Stage 2—enzymolysis: Temp. 50 °C Time ≥ 20 min pH 4	Yield of total polyphenols 30.9 g GAE/kg and yield of total anthocyanins was 2.0 g CGE/kg (extraction yield increased 45% and 38% in comparison with the control sample)	[63]
Helianthus annuus L. (Compositae) florets and petals	Total phenolic content	Enzymolysis: Viscozyme® L enzyme concentration 0.5% Sample–to–liquid ratio 0.15:10 (w/v) Solvent: d,l-menthol: d,l-lactic acid (M:HLac) (1:2) Solvent–to–water ratio 0.6 Time 2 h Temp. 40 °C	Polyphenol yield 2.98 mg/100 g	[65]
Juglans regia L. (Juglandaceae) seeds	Total phenolic content and iodine value	Enzymolysis: 2% Cellulase Time 47.37 min Temp. 45 ± 5 °C pH 5 Incubation with shaking: n-hexane Sample–to–liquid ratio 1:4 Time 110.91 min Temp. 56 °C	Total phenolic contents 478.34 mg GAE/kg and Iodine value 150.35 g I2/100 g oil	[61]
Malpighia emarginata DC. (Malpighiaceae) fruits	Phenolic compounds and antioxidant activity	Enzymolysis: Celluclast 1.5 L enzyme concentration of 8.1 NCU/g (0.9% v/w) 1:2 (w/w) acerola mash/water ratio; Temp. 50 °C; Time 2 h	Lower phenolics content (9.0%); lower antioxidant activity in comparison with ultrasound-assisted extraction	[78]
Mangifera indica L. (Anacardiaceae) peels	Total phenolic content	Enzyme-assisted ultrasound extraction: Alcalase Enzyme concentration 3.3% pH 5.5 Temp. 63 °C Time 110 min Ultrasonic power 90 W	Yield 33.56 ± 1.04 mg GAE/g fresh weight	[79]
Medicago sativa L. (Leguminosae) leaves	Total phenolic content content of phenolic acids and flavonoids antioxidant activity	Enzymolysis: Kemzyme (mixture of xylanase, β-glucanase, cellulase, amylase, and protease) Enzyme concentration 2.96% Temp. 38.96 °C pH 6.0 Time 58.92 min Extraction: Temp. 50 °C Pressure 216 bar 19.4% ethanol	Yield 546 ± 21 μg/g Increased elution of phenolic acids and flavonoids; enhanced antioxidant activity	[11]
Punica granatum L. (Lythraceae) peels	Total phenolic content (TPC)	Enzymolysis: Viscozyme 0.6% (v/w), Time 1 h pH 4.5 Temp. 40 °C sample–to–solvent ratio 1:20 Microwave-assisted extraction: Power 420 W 30% ethanol Time 140–150 min; Solvent–to–sample ratio 30:1	Increased TPC (>309.3 mg GAE/g)	[80]
Pinus koraiensis Siebold and Zucc. (Pinaceae) Pinus koraiensis nut-coated film	Flavonoids	Enzymolysis: Cellulase 90 U/g Solvent–to–sample ratio 20:1 (mL/g) Time 2 h pH 5 Temp. 25 °C 50% acetone	Yield 3.37%	[81]
Raphanus sativus L. (Brassicaceae) roots	Total phenolic contents, Trolox equivalent antioxidant capacity and radical scavenging activity	Enzymolysis: Enzyme cocktail including β-xylosidase, xyloglucanase, pectinases, xylanase, α-amylases, β-glucosidase, and protease Enzyme concentration 3.5% (w/w) Time 66 min Temp. 46 °C pH 6.1 Extraction: 80% ethanol	Extract yield 19.2 g/100 total phenolic content 48.9 mg GAE/g Trolox equivalent antioxidant capacity 270.7 mg TE/g radical scavenging activity 14.13 mg/mL reducing power 0.486 mg/mL	[26]
Rosmarinus officinalis L. (Lamiaceae) leaves	Total phenolic content (TPC)	Enzymolysis: Alcalase 2.4 L FG, Bioprep 3000 L (Cellic CTec2, Viscozyme L and Cellic HTec2 were also tested) 2.5 g enzyme/100 g raw material Time 1 h Temp. 50 °C Solid–liquid conventional extraction: Solvent–to–sample ratio 20:1 (v/w) 50% ethanol Time 24 h Room temperature	TPC 15.2 ± 0.3 mgGAE/g (extraction efficiency increased by >30% and higher DPPH radical scavenging ability)	[64]
Rubus idaeus L. (Rosaceae) seeds/pomace	Total phenolic content antioxidant activity	Enzymolysis: 1.2 units of alkaline protease/100 g Time 2 h Temp. 60 °C pH 9	Yield of polyphenols 3.7 g/100 g total ellagic acid 1.46 g/100 g antioxidant activity (by 25–48%)	[67]
Scutellaria baicalensis Georgi (Lamiaceae) plant	Baicalin	Enzymolysis: Endophytic cellulase; enzyme dose 20 U/mL Time 24 h pH 7.0 Temp. 37 °C Extraction: reflux extraction Time 3 h sample–to–solvent ratio 1:9 80% ethanol	Yield 1.56 ± 0.01 g/5 g (extraction yield increased by 79.31% in comparison with the traditional extraction method)	[82]
Solanum lycopersicum L. (Solanaceae) industrial tomato waste (peel and seed)	Total phenolic content; antioxidant activity	Enzymolysis: Celluclast:Pectinex 1:1 Enzyme: substrate ratio 0.2 mL/g Time 5 h Temp. 40 °C pH = 4.5 Extraction: Ethyl acetate extraction Solvent–to–substrate ratio 5 mL/g Time 1 h	Higher phenolic concentration; improved antioxidant properties	[71]
Ziziphus jujuba Mill. (Rhamnaceae) peel	Content of polyphenols and flavonoids Colour measurement	Enzymolysis: Mixtue of cellulase, pectinase, and protease 6000 U:2900 U:3300 U ratio per sample gram 16 mL buffer liquid volume; pH 7.0 Temp. 43 °C; Time 97 min	Yield of total polyphenols 9.02 ± 0.63 mg/g yield of total flavonoids 11.14 ± 0.45 mg/g pigment 8.93 ± 0.43	[83]
Oils and fatty acids
Arthrospira platensis (Microcoleaceae)	Oil content	Enzymolysis: 2% v/w Vinoflow® (β-glucanase (exo-1,3-) preparation) Time 24 h Temp. 40 °C pH 6.5 Alcalase®	Highest oil recovery 8.10 ± 0.20% (w/w) and increased amount of unsaturated fatty acids The most effective destruction of cell integrity and the highest extraction yield of hydrophilic biocomponents	[54]
Camellia oleifera Abel (Theaceae) seed	Oil yield and physicochemical properties	Enzymolysis: 1% (v/w) protease and 1% (v/w) cellulase) Sample–to–liquid ratio 1:6 Time 6 h Temp. 50 °C pH 5.0 Demulsification: 20% ethanol (v/v)	Free oil yield 91.38% Higher vitamin E and squalene content	[59]
Rubus idaeus L. (Rosaceae) seed/pomace	Lipophilic compounds and oil recovery	Enzymolysis: 1.2 units alkaline protease/100 g in aqueous medium Time 2 h Temp. 60 °C pH = 9	Yield 2.2% (lower than obtained with the organic solvent extraction)	[67]
Sesamum indicum L. (Pedaliaceae) seeds	Total oil content	Enzymolysis: Pectinase, protease, and a mixture of α-amylase and amylo-glucosidase (1:1 ratio) 5% of each of the three enzymes (by seed weight) pH 4.0, 6.8 or 6.8 Temp. 40 °C Time 60 min Three-phase partitioning: 40% ammonium sulphate Slurry/t-butanol ratio 1:1 pH 5.0	Highest oil recovery 86.12%	[66]
Strobilanthes crispus L. [Sericocalyx crispus (L.) Bremek]2 (Acanthaceae) leaves	Compounds with anti-hypercholesteromic activity: hexadecanoic acid, octadecanoic acid, squalene extract yield %	Enzymolysis: Cellulase 70 mg/g Time 2 h pH 4–6 Solvent–to–sample ratio 20:1 (v/w) Ultrasound-assisted extraction: 50% ethanol Time 1 h Temp. 30 °C	Yield 48.63%	[84]
Sugars and polysaccharides
Annona squamosa L. (Annonaceae) fruit	Total reducing sugar	Enzymolysis: Pectinase Enzyme concentration 2.21% Temp. 47 °C Time 4.47 h pH 4.9–4.36	Total reducing sugars 32.16 ± 0.77 mg GE/mL	[58]
Daucus carota L. (Apiaceae) roots	Total carbohydrates	Enzymolysis: Hemicellulase Time 5 h Temp. 40 °C pH 5.2 High power ultrasound-pretreatment: Ultrasonic power 12.27 W/cm2 20 kHz, 80% amplitude, Time 20 min pH 5.2	Total carbohydrate content 97.0 ± 3.0% (w/w)	[85]
Hedyotis corymbosa L. [Oldenlandia corymbosa L.] 2 (Rubiaceae)	Polysaccharides	Enzymolysis: Cellulase, Enzyme concentration 3% Solvent–to–sample ratio 30:1 Time 10 min pH 5 Temp. 56 °C Ultrasonic power 200 W	Yield 4.10 ± 0.16%	[86]
Helianthus annuus L. (Compositae) florets and petals	Total amount of reducing sugars	Enzymolysis: Viscozyme® L enzyme concentration 0.5% Sample–to–liquid ratio 0.15:10 (w/v) Solvent: d,l-menthol:d,l-lactic acid (M:HLac) (1:2) Solvent–to–water ratio 0.6 Time 2 h Temp. 40 °C	Yield of reducing sugars 20.92 mg/100 g	[65]
Lycium barbarum L. (Solanaceae) fruit	Polysaccharides	Enzymolysis: 2.15% Cellulase Time 20 min Temp. 56 °C pH 4.6 Ultrasonic power 80 W	Yield 6.31 ± 0.03%	[87]
Morinda officinalis F.C.How (Rubiaceae) radix	Polysaccharides and radical scavenging activity	Enzymolysis: 1.0% cellulase, 1.5% pectinase, 1.0% papain Solvent–to–sample ratio 21 mL/g pH 5.3 Temp. 50 °C Time 60 min Ultrasonic power 280 W	Yield 23.68% ± 0.52% and DPPH radical scavenging activity 117.26 mg vitamin C equivalents/100 g	[69]
Mytilus coruscus (Mytilidae) mussel meat	Polysaccharide	Enzymolysis: Acid protease Enzyme concentration 3.2% Solvent–to–sample ratio 30:1 (mL/g) Time 36 min Temperature 64 °C pH 3.0 Ultrasonic power 60 W	Yield 12.86 ± 0.12%	[6]
Rosa roxburghii Tratt. (Rosaceae) fruits	Yield of polysaccharides	Enzymolysis: Solvent–to–sample ratio 13.5:1 mL/g Cellulase concentration 6.5 g/mL Microwave power 575 W Time 18 min	Yield of 36.21 ± 0.62%	[88]
Schisandra chinensis (Turcz.) Baill. (Schisandraceae) fruit	Polysaccharides	Enzymolysis: Enzyme complex papain/pectinase/cellulase 1:1:1 enzyme concentration 1.5% Microwave irradiation time 10 min Time 3 h Temp. 48 °C pH 4.2	Yield 7.38 ± 0.21%	[74]
Tuber aestivum Vittad. (Tuberaceae) fruiting body	Polysaccharides	Enzymolysis: Mixture of 1% trypsin, 1% papain, and 2% pectinase Time 90 min Temp. 50 °C pH 6.0	Yield 46.93% of total polysaccharide and 46.5 ± 2.29 mg/g of uronic acids	[56]
Viscum coloratum (Kom.) Nakai (Santalaceae) leaves	Yield of polysaccharides, free radical scavenging and hydroxyl radical scavenging activity	Enzymolysis: Rehydration (1:20, w/v; 30 min; 45 °C) Cellulase concentration 2.5% Sample–to–solvent ratio 1:40 Time 40 min Temp. 50 °C pH of 5.0	Yield 21.83 ± 0.45%, DPPH Radical scavenging 80.01 ± 2.31% and the hydroxyl radical scavenging 38.26 ± 1.79%	[68]
Proteins
Olea europaea L. (Oleaceae) leaves	Total protein amount	Enzymolysis: Cellulase (Celluclast 1.5 L) Enzyme concentration 5% (v/v) pH 5.0 Temp. 55 °C Time 15 min 30% acetonitrile	Total protein amount 1.87–6.64 mg/g. These contents were higher (ca. 2–3 times) than those found using other extraction protocols	[12]
Sesamum indicum L. (Pedaliaceae)	Protein	Enzymolysis: Alcalase 1.94 AU/100 g enzyme concentration microwave extractor Temp. 49 °C Time 98 min pH 9.8 Alkaline extraction: pH 9.5 Temp. 45 °C Time 30 min	Protein yield 76.6% (higher than without enzyme pretreatment)	[52]
Soy pulp (okara, a byproduct from soymilk processing)	Protein	Viscozyme 4% Temp. 53 °C pH 6.2, Time 2 h	Yield of protein 56% (dry weight basis), recovery of 28% (increased in comparison to the sample with no enzymatic pretreatment)	[10]
Essential oil
Citrus sinensis L. Osbeck (Rutaceae) peel	Essential oil	Enzymolysis: Viscozyme L 3.9 mL/100 g Solvent–to–sample ratio 4.0 (mL/g) Time 3.8 h Temp. 55 °C pH 5.5	Yield 46.31 ± 0.32 mL/kg	[89]
Forsythia suspensa (Thunb.) Vahl (Oleaceae) fruit	Essential oil	Enzymolysis: Cellulase concentration 0.5% (w/w) Sample–to–liquid ratio 1:10 Time 25 min Temp. 40 °C pH 5.0 Irradiation power 500 W	Yield 3.27% (increased by 39.15–45.33%)	[60]
Coriandrum sativum L. (Apiaceae) seeds	Total lipid content, fatty acid profiles, and lipid quality	Enzymolysis: Cellulase (10 mg/100 g) Time 1 h Temp. 40 °C Hydrodistillation in Clevenger-type apparatus; Time 2 h	Improved essential oil yield (by 33.3–44.2%) and its main component linalool increased amount of oxygenated terpenes	[13]
Diterpenes and triterpenes
Glycyrrhiza glabra L. (Leguminosae) root	Glycyrrhizic acid Total liquorice extraction yield	Enzymolysis: Cellulase or hemicellulase (2% w/v) or Multizyme® Cellulase CEP concentrate (3% w/v) pH 5.0 Temp. 45 °C Time 1 h; Reflux or ultrasound - assisted extraction	Increased total liquorice (by 42.77–44.95%) and glycyrrhizic acid extraction yield	[62]
Pseuderanthemum palatiferum (Nees) Radlk. [Pseuderanthemum latifolium B. Hansen] 2 (Acanthaceae) leaf	Triterpenoid saponins	Enzymolysis: Viscozyme 7.5 μL/g, Water–to–material ratio of 12.5 mL/g Temp. 54.9 °C Time 80.8 min	Yield of 64.19%, (significantly higher than that in Soxhlet extraction)	[14]
Stevia rebaudiana (Bertoni) Bertoni (Compositae) leaves	Stevioside (diterpene glycoside)	Enzymolysis: Hemicellulase at 60 °C or Cellulase at 50 °C pH 5.0 Time 45–60 min	High stevioside yield (369.23 ± 0.11 μg)	[70]
Carotenoids
Daucus carota L. (Apiaceae)	Carotenoids	Enzymolysis: Fructozym® MA concentration 0.3 mL/100 g Time 24 h Temp. 37 °C pH 7.4	Yield 393.4 μg/mL	[90]
Daucus carota subsp. sativus (Hoffm.) Arcang (Apiaceae) carrot pomace powder	β-carotene	Enzymolysis: Endozym Pectofruit (commercial pectinase) 61 U/mL 5 mL of enzyme/100 mg of plant material Time 60 min Temp. 45 °C pH = 5.5	Yield 0.85 ± 0.11 mg/g (improved extraction efficiency up to 90%)	[15]
Haematococcus pluvialis (Haematococcaceae)	Astaxanthin	Enzymolysis: Pectinase enzyme content 0.08% Time 3 h Temp. 55 °C pH = 4.5	Extract yield 75.30%	[53]
Helianthus annuus L. (Compositae) florets and petals	Total amount of carotenoids	Enzymolysis: Viscozyme® L enzyme concentration 0.5% Sample–to–liquid ratio 0.15:10 (w/v) Solvent: d,l-menthol:d,l-lactic acid (M:HLac) (1:2) Solvent–to–water ratio 0.6 Time 2 h Temp. 40 °C	Yield of carotenoids 1449 mg/100 g	[65]
Solanum lycopersicum L. (Solanaceae) industrial tomato waste (peel and seeds)	Lycopene	Enzymolysis: Celluclast:Pectinex 1:1 Enzyme: substrate ratio 0.2 mL/g Time 5 h Temp. 40 °C pH = 4.5 Extraction: Ethyl acetate extraction Solvent–substrate ratio 5 mL/g Time 1 h	Higher lycopene recovery	[71]
Others
Beta vulgaris L. (Amaranthaceae) beetroot	Betalains betacyanin, and betaxanthin	Enzymolysis: Enzymatic mix 25 U/g containing cellulase (37%), xylanase (35%), pectinase (28%) Temp. 25 °C Time 240 min pH 5.5 ± 0.1	Yield (mg/mL U) betaxanthin 11.37 ± 0.45, betacyanin 14.67 ± 0.67	[72]
Malpighia emarginata DC. (Malpighiaceae) fruit	Ascorbic acid	Enzymolysis: Celluclast 1.5 L enzyme concentration of 8.1 NCU/g (0.9% v/w) 1:2 (w/w) acerola mash/water ratio Temp. 50 °C Time 2 h	Lower content of vitamin C (35.7%) in comparison with ultrasound-assisted extraction	[78]
Piper nigrum L. (Piperaceae)	Piperine	Enzymolysis: 0.08% cellulase, 0.1% neutral protease, 0.4% surfactant (sodium stearoyl lactylate) (w/w) Sample–to–liquid ratio 1:5 (g/mL) Temp. 60 °C Time 4 h; Granularity less than 10 mesh	Content of piperine spectrophotometric method 4.54%, HPLC 4.42%	[51]
Zingiber officinale Roscoe (Cochin variety) (Zingiberaceae) rhizome	Oleoresin yield and 6-gingerol content	Enzymolysis: Cellulase, pectinase, α-amylase, and Viscozyme concentration 0.5% (mL/w) pH 4.5–5.0 Temp. 50 °C Time 30–120 min Protease concentration 0.5 g/g pH 6.0; Temp. 55 °C Extraction: multistage extraction with ethanol and acetone in glass columns	Improved yield of resin (20–21%) and content of gingerol in resins (10.1–12.2%)	[16]

¹ metabolites, product, or activity; ² currently accepted botanical name according to www.theplantlist.org; accessed on 10 January 2022.

Table 1. Examples of studies on enzyme-assisted extraction of metabolites from natural materials (plants, mushrooms, and animals).

Natural Material	Target 1	Optimized Conditions of Enzymolysis and Extraction	Extraction Effects and Yields in Optimal Conditions	Ref.
Polyphenols and phenols
Annona squamosa L. (Annonaceae) fruits	Yield and quality of juice; total phenolic content	Pectinase Enzyme concentration 2.21% Temp. 47 °C Time 4.47 h pH 4.9–4.36	Yield 88% (w/w) Total polyphenols 93.95 ± 0.51 μg of GAE/mL	[58]
Coriandrum sativum L. (Apiaceae) residue from the seed distillation process	Total phenolic (TPC) and total flavonoid (TFC) contents antioxidant activity	Enzymolysis: Cellulase (10 mg/100 g) Time 1 h Temp. 40 °C Hydrodistillation in Clevenger-type apparatus Time 2 h	Increased release of phenolic compounds improved antioxidant activity	[13]
Crocus sativus L. (Iridaceae) tepals	Total phenolic content and total anthocyanin content	Enzymolysis: Mixture of cellulolytic and hemicellulolytic preparations (1:1) Enzyme concentration: 0.12–0.15% Solvent–to–sample ratio 10:1 (v/w) Time 145–185 min Stage 1—rehydration: Time 1 h pH 4.0 Temp. 25 °C Stage 2—enzymolysis: Temp. 50 °C Time ≥ 20 min pH 4	Yield of total polyphenols 30.9 g GAE/kg and yield of total anthocyanins was 2.0 g CGE/kg (extraction yield increased 45% and 38% in comparison with the control sample)	[63]
Helianthus annuus L. (Compositae) florets and petals	Total phenolic content	Enzymolysis: Viscozyme® L enzyme concentration 0.5% Sample–to–liquid ratio 0.15:10 (w/v) Solvent: d,l-menthol: d,l-lactic acid (M:HLac) (1:2) Solvent–to–water ratio 0.6 Time 2 h Temp. 40 °C	Polyphenol yield 2.98 mg/100 g	[65]
Juglans regia L. (Juglandaceae) seeds	Total phenolic content and iodine value	Enzymolysis: 2% Cellulase Time 47.37 min Temp. 45 ± 5 °C pH 5 Incubation with shaking: n-hexane Sample–to–liquid ratio 1:4 Time 110.91 min Temp. 56 °C	Total phenolic contents 478.34 mg GAE/kg and Iodine value 150.35 g I2/100 g oil	[61]
Malpighia emarginata DC. (Malpighiaceae) fruits	Phenolic compounds and antioxidant activity	Enzymolysis: Celluclast 1.5 L enzyme concentration of 8.1 NCU/g (0.9% v/w) 1:2 (w/w) acerola mash/water ratio; Temp. 50 °C; Time 2 h	Lower phenolics content (9.0%); lower antioxidant activity in comparison with ultrasound-assisted extraction	[78]
Mangifera indica L. (Anacardiaceae) peels	Total phenolic content	Enzyme-assisted ultrasound extraction: Alcalase Enzyme concentration 3.3% pH 5.5 Temp. 63 °C Time 110 min Ultrasonic power 90 W	Yield 33.56 ± 1.04 mg GAE/g fresh weight	[79]
Medicago sativa L. (Leguminosae) leaves	Total phenolic content content of phenolic acids and flavonoids antioxidant activity	Enzymolysis: Kemzyme (mixture of xylanase, β-glucanase, cellulase, amylase, and protease) Enzyme concentration 2.96% Temp. 38.96 °C pH 6.0 Time 58.92 min Extraction: Temp. 50 °C Pressure 216 bar 19.4% ethanol	Yield 546 ± 21 μg/g Increased elution of phenolic acids and flavonoids; enhanced antioxidant activity	[11]
Punica granatum L. (Lythraceae) peels	Total phenolic content (TPC)	Enzymolysis: Viscozyme 0.6% (v/w), Time 1 h pH 4.5 Temp. 40 °C sample–to–solvent ratio 1:20 Microwave-assisted extraction: Power 420 W 30% ethanol Time 140–150 min; Solvent–to–sample ratio 30:1	Increased TPC (>309.3 mg GAE/g)	[80]
Pinus koraiensis Siebold and Zucc. (Pinaceae) Pinus koraiensis nut-coated film	Flavonoids	Enzymolysis: Cellulase 90 U/g Solvent–to–sample ratio 20:1 (mL/g) Time 2 h pH 5 Temp. 25 °C 50% acetone	Yield 3.37%	[81]
Raphanus sativus L. (Brassicaceae) roots	Total phenolic contents, Trolox equivalent antioxidant capacity and radical scavenging activity	Enzymolysis: Enzyme cocktail including β-xylosidase, xyloglucanase, pectinases, xylanase, α-amylases, β-glucosidase, and protease Enzyme concentration 3.5% (w/w) Time 66 min Temp. 46 °C pH 6.1 Extraction: 80% ethanol	Extract yield 19.2 g/100 total phenolic content 48.9 mg GAE/g Trolox equivalent antioxidant capacity 270.7 mg TE/g radical scavenging activity 14.13 mg/mL reducing power 0.486 mg/mL	[26]
Rosmarinus officinalis L. (Lamiaceae) leaves	Total phenolic content (TPC)	Enzymolysis: Alcalase 2.4 L FG, Bioprep 3000 L (Cellic CTec2, Viscozyme L and Cellic HTec2 were also tested) 2.5 g enzyme/100 g raw material Time 1 h Temp. 50 °C Solid–liquid conventional extraction: Solvent–to–sample ratio 20:1 (v/w) 50% ethanol Time 24 h Room temperature	TPC 15.2 ± 0.3 mgGAE/g (extraction efficiency increased by >30% and higher DPPH radical scavenging ability)	[64]
Rubus idaeus L. (Rosaceae) seeds/pomace	Total phenolic content antioxidant activity	Enzymolysis: 1.2 units of alkaline protease/100 g Time 2 h Temp. 60 °C pH 9	Yield of polyphenols 3.7 g/100 g total ellagic acid 1.46 g/100 g antioxidant activity (by 25–48%)	[67]
Scutellaria baicalensis Georgi (Lamiaceae) plant	Baicalin	Enzymolysis: Endophytic cellulase; enzyme dose 20 U/mL Time 24 h pH 7.0 Temp. 37 °C Extraction: reflux extraction Time 3 h sample–to–solvent ratio 1:9 80% ethanol	Yield 1.56 ± 0.01 g/5 g (extraction yield increased by 79.31% in comparison with the traditional extraction method)	[82]
Solanum lycopersicum L. (Solanaceae) industrial tomato waste (peel and seed)	Total phenolic content; antioxidant activity	Enzymolysis: Celluclast:Pectinex 1:1 Enzyme: substrate ratio 0.2 mL/g Time 5 h Temp. 40 °C pH = 4.5 Extraction: Ethyl acetate extraction Solvent–to–substrate ratio 5 mL/g Time 1 h	Higher phenolic concentration; improved antioxidant properties	[71]
Ziziphus jujuba Mill. (Rhamnaceae) peel	Content of polyphenols and flavonoids Colour measurement	Enzymolysis: Mixtue of cellulase, pectinase, and protease 6000 U:2900 U:3300 U ratio per sample gram 16 mL buffer liquid volume; pH 7.0 Temp. 43 °C; Time 97 min	Yield of total polyphenols 9.02 ± 0.63 mg/g yield of total flavonoids 11.14 ± 0.45 mg/g pigment 8.93 ± 0.43	[83]
Oils and fatty acids
Arthrospira platensis (Microcoleaceae)	Oil content	Enzymolysis: 2% v/w Vinoflow® (β-glucanase (exo-1,3-) preparation) Time 24 h Temp. 40 °C pH 6.5 Alcalase®	Highest oil recovery 8.10 ± 0.20% (w/w) and increased amount of unsaturated fatty acids The most effective destruction of cell integrity and the highest extraction yield of hydrophilic biocomponents	[54]
Camellia oleifera Abel (Theaceae) seed	Oil yield and physicochemical properties	Enzymolysis: 1% (v/w) protease and 1% (v/w) cellulase) Sample–to–liquid ratio 1:6 Time 6 h Temp. 50 °C pH 5.0 Demulsification: 20% ethanol (v/v)	Free oil yield 91.38% Higher vitamin E and squalene content	[59]
Rubus idaeus L. (Rosaceae) seed/pomace	Lipophilic compounds and oil recovery	Enzymolysis: 1.2 units alkaline protease/100 g in aqueous medium Time 2 h Temp. 60 °C pH = 9	Yield 2.2% (lower than obtained with the organic solvent extraction)	[67]
Sesamum indicum L. (Pedaliaceae) seeds	Total oil content	Enzymolysis: Pectinase, protease, and a mixture of α-amylase and amylo-glucosidase (1:1 ratio) 5% of each of the three enzymes (by seed weight) pH 4.0, 6.8 or 6.8 Temp. 40 °C Time 60 min Three-phase partitioning: 40% ammonium sulphate Slurry/t-butanol ratio 1:1 pH 5.0	Highest oil recovery 86.12%	[66]
Strobilanthes crispus L. [Sericocalyx crispus (L.) Bremek]2 (Acanthaceae) leaves	Compounds with anti-hypercholesteromic activity: hexadecanoic acid, octadecanoic acid, squalene extract yield %	Enzymolysis: Cellulase 70 mg/g Time 2 h pH 4–6 Solvent–to–sample ratio 20:1 (v/w) Ultrasound-assisted extraction: 50% ethanol Time 1 h Temp. 30 °C	Yield 48.63%	[84]
Sugars and polysaccharides
Annona squamosa L. (Annonaceae) fruit	Total reducing sugar	Enzymolysis: Pectinase Enzyme concentration 2.21% Temp. 47 °C Time 4.47 h pH 4.9–4.36	Total reducing sugars 32.16 ± 0.77 mg GE/mL	[58]
Daucus carota L. (Apiaceae) roots	Total carbohydrates	Enzymolysis: Hemicellulase Time 5 h Temp. 40 °C pH 5.2 High power ultrasound-pretreatment: Ultrasonic power 12.27 W/cm2 20 kHz, 80% amplitude, Time 20 min pH 5.2	Total carbohydrate content 97.0 ± 3.0% (w/w)	[85]
Hedyotis corymbosa L. [Oldenlandia corymbosa L.] 2 (Rubiaceae)	Polysaccharides	Enzymolysis: Cellulase, Enzyme concentration 3% Solvent–to–sample ratio 30:1 Time 10 min pH 5 Temp. 56 °C Ultrasonic power 200 W	Yield 4.10 ± 0.16%	[86]
Helianthus annuus L. (Compositae) florets and petals	Total amount of reducing sugars	Enzymolysis: Viscozyme® L enzyme concentration 0.5% Sample–to–liquid ratio 0.15:10 (w/v) Solvent: d,l-menthol:d,l-lactic acid (M:HLac) (1:2) Solvent–to–water ratio 0.6 Time 2 h Temp. 40 °C	Yield of reducing sugars 20.92 mg/100 g	[65]
Lycium barbarum L. (Solanaceae) fruit	Polysaccharides	Enzymolysis: 2.15% Cellulase Time 20 min Temp. 56 °C pH 4.6 Ultrasonic power 80 W	Yield 6.31 ± 0.03%	[87]
Morinda officinalis F.C.How (Rubiaceae) radix	Polysaccharides and radical scavenging activity	Enzymolysis: 1.0% cellulase, 1.5% pectinase, 1.0% papain Solvent–to–sample ratio 21 mL/g pH 5.3 Temp. 50 °C Time 60 min Ultrasonic power 280 W	Yield 23.68% ± 0.52% and DPPH radical scavenging activity 117.26 mg vitamin C equivalents/100 g	[69]
Mytilus coruscus (Mytilidae) mussel meat	Polysaccharide	Enzymolysis: Acid protease Enzyme concentration 3.2% Solvent–to–sample ratio 30:1 (mL/g) Time 36 min Temperature 64 °C pH 3.0 Ultrasonic power 60 W	Yield 12.86 ± 0.12%	[6]
Rosa roxburghii Tratt. (Rosaceae) fruits	Yield of polysaccharides	Enzymolysis: Solvent–to–sample ratio 13.5:1 mL/g Cellulase concentration 6.5 g/mL Microwave power 575 W Time 18 min	Yield of 36.21 ± 0.62%	[88]
Schisandra chinensis (Turcz.) Baill. (Schisandraceae) fruit	Polysaccharides	Enzymolysis: Enzyme complex papain/pectinase/cellulase 1:1:1 enzyme concentration 1.5% Microwave irradiation time 10 min Time 3 h Temp. 48 °C pH 4.2	Yield 7.38 ± 0.21%	[74]
Tuber aestivum Vittad. (Tuberaceae) fruiting body	Polysaccharides	Enzymolysis: Mixture of 1% trypsin, 1% papain, and 2% pectinase Time 90 min Temp. 50 °C pH 6.0	Yield 46.93% of total polysaccharide and 46.5 ± 2.29 mg/g of uronic acids	[56]
Viscum coloratum (Kom.) Nakai (Santalaceae) leaves	Yield of polysaccharides, free radical scavenging and hydroxyl radical scavenging activity	Enzymolysis: Rehydration (1:20, w/v; 30 min; 45 °C) Cellulase concentration 2.5% Sample–to–solvent ratio 1:40 Time 40 min Temp. 50 °C pH of 5.0	Yield 21.83 ± 0.45%, DPPH Radical scavenging 80.01 ± 2.31% and the hydroxyl radical scavenging 38.26 ± 1.79%	[68]
Proteins
Olea europaea L. (Oleaceae) leaves	Total protein amount	Enzymolysis: Cellulase (Celluclast 1.5 L) Enzyme concentration 5% (v/v) pH 5.0 Temp. 55 °C Time 15 min 30% acetonitrile	Total protein amount 1.87–6.64 mg/g. These contents were higher (ca. 2–3 times) than those found using other extraction protocols	[12]
Sesamum indicum L. (Pedaliaceae)	Protein	Enzymolysis: Alcalase 1.94 AU/100 g enzyme concentration microwave extractor Temp. 49 °C Time 98 min pH 9.8 Alkaline extraction: pH 9.5 Temp. 45 °C Time 30 min	Protein yield 76.6% (higher than without enzyme pretreatment)	[52]
Soy pulp (okara, a byproduct from soymilk processing)	Protein	Viscozyme 4% Temp. 53 °C pH 6.2, Time 2 h	Yield of protein 56% (dry weight basis), recovery of 28% (increased in comparison to the sample with no enzymatic pretreatment)	[10]
Essential oil
Citrus sinensis L. Osbeck (Rutaceae) peel	Essential oil	Enzymolysis: Viscozyme L 3.9 mL/100 g Solvent–to–sample ratio 4.0 (mL/g) Time 3.8 h Temp. 55 °C pH 5.5	Yield 46.31 ± 0.32 mL/kg	[89]
Forsythia suspensa (Thunb.) Vahl (Oleaceae) fruit	Essential oil	Enzymolysis: Cellulase concentration 0.5% (w/w) Sample–to–liquid ratio 1:10 Time 25 min Temp. 40 °C pH 5.0 Irradiation power 500 W	Yield 3.27% (increased by 39.15–45.33%)	[60]
Coriandrum sativum L. (Apiaceae) seeds	Total lipid content, fatty acid profiles, and lipid quality	Enzymolysis: Cellulase (10 mg/100 g) Time 1 h Temp. 40 °C Hydrodistillation in Clevenger-type apparatus; Time 2 h	Improved essential oil yield (by 33.3–44.2%) and its main component linalool increased amount of oxygenated terpenes	[13]
Diterpenes and triterpenes
Glycyrrhiza glabra L. (Leguminosae) root	Glycyrrhizic acid Total liquorice extraction yield	Enzymolysis: Cellulase or hemicellulase (2% w/v) or Multizyme® Cellulase CEP concentrate (3% w/v) pH 5.0 Temp. 45 °C Time 1 h; Reflux or ultrasound - assisted extraction	Increased total liquorice (by 42.77–44.95%) and glycyrrhizic acid extraction yield	[62]
Pseuderanthemum palatiferum (Nees) Radlk. [Pseuderanthemum latifolium B. Hansen] 2 (Acanthaceae) leaf	Triterpenoid saponins	Enzymolysis: Viscozyme 7.5 μL/g, Water–to–material ratio of 12.5 mL/g Temp. 54.9 °C Time 80.8 min	Yield of 64.19%, (significantly higher than that in Soxhlet extraction)	[14]
Stevia rebaudiana (Bertoni) Bertoni (Compositae) leaves	Stevioside (diterpene glycoside)	Enzymolysis: Hemicellulase at 60 °C or Cellulase at 50 °C pH 5.0 Time 45–60 min	High stevioside yield (369.23 ± 0.11 μg)	[70]
Carotenoids
Daucus carota L. (Apiaceae)	Carotenoids	Enzymolysis: Fructozym® MA concentration 0.3 mL/100 g Time 24 h Temp. 37 °C pH 7.4	Yield 393.4 μg/mL	[90]
Daucus carota subsp. sativus (Hoffm.) Arcang (Apiaceae) carrot pomace powder	β-carotene	Enzymolysis: Endozym Pectofruit (commercial pectinase) 61 U/mL 5 mL of enzyme/100 mg of plant material Time 60 min Temp. 45 °C pH = 5.5	Yield 0.85 ± 0.11 mg/g (improved extraction efficiency up to 90%)	[15]
Haematococcus pluvialis (Haematococcaceae)	Astaxanthin	Enzymolysis: Pectinase enzyme content 0.08% Time 3 h Temp. 55 °C pH = 4.5	Extract yield 75.30%	[53]
Helianthus annuus L. (Compositae) florets and petals	Total amount of carotenoids	Enzymolysis: Viscozyme® L enzyme concentration 0.5% Sample–to–liquid ratio 0.15:10 (w/v) Solvent: d,l-menthol:d,l-lactic acid (M:HLac) (1:2) Solvent–to–water ratio 0.6 Time 2 h Temp. 40 °C	Yield of carotenoids 1449 mg/100 g	[65]
Solanum lycopersicum L. (Solanaceae) industrial tomato waste (peel and seeds)	Lycopene	Enzymolysis: Celluclast:Pectinex 1:1 Enzyme: substrate ratio 0.2 mL/g Time 5 h Temp. 40 °C pH = 4.5 Extraction: Ethyl acetate extraction Solvent–substrate ratio 5 mL/g Time 1 h	Higher lycopene recovery	[71]
Others
Beta vulgaris L. (Amaranthaceae) beetroot	Betalains betacyanin, and betaxanthin	Enzymolysis: Enzymatic mix 25 U/g containing cellulase (37%), xylanase (35%), pectinase (28%) Temp. 25 °C Time 240 min pH 5.5 ± 0.1	Yield (mg/mL U) betaxanthin 11.37 ± 0.45, betacyanin 14.67 ± 0.67	[72]
Malpighia emarginata DC. (Malpighiaceae) fruit	Ascorbic acid	Enzymolysis: Celluclast 1.5 L enzyme concentration of 8.1 NCU/g (0.9% v/w) 1:2 (w/w) acerola mash/water ratio Temp. 50 °C Time 2 h	Lower content of vitamin C (35.7%) in comparison with ultrasound-assisted extraction	[78]
Piper nigrum L. (Piperaceae)	Piperine	Enzymolysis: 0.08% cellulase, 0.1% neutral protease, 0.4% surfactant (sodium stearoyl lactylate) (w/w) Sample–to–liquid ratio 1:5 (g/mL) Temp. 60 °C Time 4 h; Granularity less than 10 mesh	Content of piperine spectrophotometric method 4.54%, HPLC 4.42%	[51]
Zingiber officinale Roscoe (Cochin variety) (Zingiberaceae) rhizome	Oleoresin yield and 6-gingerol content	Enzymolysis: Cellulase, pectinase, α-amylase, and Viscozyme concentration 0.5% (mL/w) pH 4.5–5.0 Temp. 50 °C Time 30–120 min Protease concentration 0.5 g/g pH 6.0; Temp. 55 °C Extraction: multistage extraction with ethanol and acetone in glass columns	Improved yield of resin (20–21%) and content of gingerol in resins (10.1–12.2%)	[16]

¹ metabolites, product, or activity; ² currently accepted botanical name according to www.theplantlist.org; accessed on 10 January 2022.

4. Conclusions

Enzyme-assisted extraction is a developing technique, which seems to be a promising tool for more efficient exploitation of natural materials. It is an attractive alternative to conventional extraction techniques since it is a sustainable and eco-friendly technology. Many studies have shown its highly positive effects in improvement of the yield of different classes of bioactive metabolites (e.g., polyphenols, carotenoids, proteins, polysaccharides, terpenes, and lipids) for nutraceutical and pharmaceutical applications. EAE facilitates the use of more mild extraction conditions (e.g., lower temperatures), does not require application of highly specialized and expensive equipment, and allows reducing the consumption of toxic solvents. It enables additional recovery of compounds from agricultural wastes. However, there are no universal conditions for achievement of satisfactory results with the use of this technique. Process optimization adapted to the material (enzyme type, particle size, hydration etc.), metabolites, and enzyme used (e.g., temperature, concentration, pH, and duration) is essential for efficient application of EAE. However, this requirement is quite common in other extraction techniques and does not seem to be a drawback.