## 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; 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; 57; 52]. 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) [58; 59; 14; 52; 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; 58; 61; 59; 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 \(\beta\)-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 palaliferum_ leaves [14; 62]. It was also a promising tool for elution of stevisoside 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].