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Review

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

by
Agnieszka Łubek-Nguyen
,
Wojciech Ziemichód
and
Marta Olech
*
Department of Pharmaceutical Botany, Medical University of Lublin, 1 Chodźki Street, 20-093 Lublin, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(7), 3232; https://doi.org/10.3390/app12073232
Submission received: 10 March 2022 / Revised: 17 March 2022 / Accepted: 19 March 2022 / Published: 22 March 2022
(This article belongs to the Special Issue Frontier Research in Food Processing Technologies)

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).
Table 1. Examples of studies on enzyme-assisted extraction of metabolites from natural materials (plants, mushrooms, and animals).
Natural MaterialTarget 1Optimized
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 activityEnzymolysis:
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 contentEnzyme-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
FlavonoidsEnzymolysis:
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
BaicalinEnzymolysis:
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 activityEnzymolysis:
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 recoveryEnzymolysis:
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 contentEnzymolysis:
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 sugarEnzymolysis:
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 carbohydratesEnzymolysis:
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)
PolysaccharidesEnzymolysis:
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
PolysaccharidesEnzymolysis:
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
PolysaccharideEnzymolysis:
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
PolysaccharidesEnzymolysis:
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
PolysaccharidesEnzymolysis:
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)
ProteinEnzymolysis:
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)
ProteinViscozyme 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 oilEnzymolysis:
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 oilEnzymolysis:
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)
CarotenoidsEnzymolysis:
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-caroteneEnzymolysis:
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)
AstaxanthinEnzymolysis:
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)
LycopeneEnzymolysis:
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 acidEnzymolysis:
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)
PiperineEnzymolysis:
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 contentEnzymolysis:
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.

Author Contributions

Conceptualization, M.O.; writing—original draft preparation, A.Ł.-N., W.Z. and M.O.; writing—review and editing, M.O.; visualization, M.O.; supervision, M.O. All authors have read and agreed to the published version of the manuscript.

Funding

The APC was funded by Medical University of Lublin.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The graphical abstract was created with BioRender.com, accessed on 18 January 2022.

Conflicts of Interest

The authors declare no conflict of interest.

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Łubek-Nguyen, A.; Ziemichód, W.; Olech, M. Application of Enzyme-Assisted Extraction for the Recovery of Natural Bioactive Compounds for Nutraceutical and Pharmaceutical Applications. Appl. Sci. 2022, 12, 3232. https://doi.org/10.3390/app12073232

AMA Style

Łubek-Nguyen A, Ziemichód W, Olech M. Application of Enzyme-Assisted Extraction for the Recovery of Natural Bioactive Compounds for Nutraceutical and Pharmaceutical Applications. Applied Sciences. 2022; 12(7):3232. https://doi.org/10.3390/app12073232

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Łubek-Nguyen, Agnieszka, Wojciech Ziemichód, and Marta Olech. 2022. "Application of Enzyme-Assisted Extraction for the Recovery of Natural Bioactive Compounds for Nutraceutical and Pharmaceutical Applications" Applied Sciences 12, no. 7: 3232. https://doi.org/10.3390/app12073232

APA Style

Łubek-Nguyen, A., Ziemichód, W., & Olech, M. (2022). Application of Enzyme-Assisted Extraction for the Recovery of Natural Bioactive Compounds for Nutraceutical and Pharmaceutical Applications. Applied Sciences, 12(7), 3232. https://doi.org/10.3390/app12073232

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