With the development of technology, the food market now mostly supplies processed foods to serve the consumer’s convenience. Processing, however, cannot only be used to prepare food for the consumer benefit, but also to save costs on raw materials and to replace expensive natural components by cheaper synthetic ones. Sometimes valuable components are also degraded or lost by harsh processing methods or conditions. Vitamins, minerals, amino acids and polyunsaturated fatty acids are key components of a balanced diet that prevent the deterioration of health and decrease the incidence of obesity, diabetes, cardiovascular disease and cancer. Vegetable oils are an especially valuable source of biologically active substances with useful properties in the treatment and prevention of many diseases (1-4). These substances include natural antioxidants, ω-6 and ω-3 fatty acids, which are represented by linoleic and linolenic fatty acids, respectively, as well as phytosterols and squalene – a precursor to the formation of sterols, steroid hormones and vitamin D (1-6).

The content of these substances in oil depends on the raw materials and the oil extraction process. The two most common techniques to extract oil from oilseeds are mechanical extraction by using a press (pressing) and oil extraction by using an organic solvent. Although solvent extraction is more efficient and allows to extract more oil from the raw material than pressing, this technology involves the use of organic solvents (mostly hexane), which is dangerous in the production process (flammable and explosive) and a risk for human health if not properly removed from the product (3, 7, 8). In addition, the release of solvent vapours into the environment is also hazardous from an ecological point of view, as they react with air pollutants and form ozone and photochemicals (7). Finally, the oil obtained by the extraction method must be refined, which causes the loss of many biologically active substances.

The pressing method is more environmentally friendly (despite its high energy consumption) and safe and it also helps to preserve valuable natural components in the resulting oils (especially cold pressing). However, pressing does not allow to extract oil from the oilseeds completely and a significant percentage remains in the cake. This is the reason of the ongoing research to intensify mechanical oil extraction technologies (3). Some intensification approaches include solvent extraction methods that use terpenes and ionic liquids as green solvents, but the most promising among these methods is the enzymatic pretreatment of plant materials and especially oilseeds with hydrolases before pressing (3, 4, 7, 9-11).

The oil within oilseeds is very well protected by compartimentalization and various structural components within the oilseed cells. In order to release the oil from its subcellular and cellular compartments, it is necessary to degrade these structural components. To support mechanical force that by itself does not result in a complete extraction, the use of hydrolytic enzymes, which are fast and specific catalysts, is promising to achieve a high oil yield by partial hydrolysis of various oil material cell constituents.

The lipid bodies, which contain the oil in the seed cells, are called oleosomes (8, 12-16). Oleosomes are droplets formed by triacylglycerides and surrounded by a membrane consisting of a monolayer of phospholipids and a surface protein layer that penetrates the phospholipid layer and reaches into the oleosome (15, 16). The phospholipid layer is formed mainly by phosphatidylcholine and phosphatidylserine, while oleosin predominates in the protein layer together within lower amounts of kaleosin and steroleosin (14, 15).

The structure of the plant cell wall is formed by linear cellulose chains and branched hemicellulose chains immersed in a lignin matrix and features cross-linking lignin–carbohydrate bridges, ether, and C–C bonds (17). The cell walls of oilseeds consist mainly of polysaccharides such as cellulose, hemicelluloses, lignin, pectins and arabinogalactans, but also contain a low amount of hydroxyproline-rich glycoproteins (12, 18). Cellulose is a linear chain of β-1,4-linked d-glucose units, which can be cleaved by various cellulases (Table 1 (17-41)). Hemicelluloses are linear or branched homo- or heteropolysaccharides bound to cellulose microfibrils by hydrogen bonds or connected to lignin by covalent bonds and thereby form the complex and solid structure of plant cell walls.

Hemicelluloses consist of a large number of different mono- and oligosaccharides, including glucans, xylans, mannans, galactans, xyloglucans, arabinogalactans, etc. Based on their composition, arabinogalactans belong to the hemicelluloses. Despite not containing galacturonic acid residues, some authors also refer to arabinogalactans as pectins, due to their presence in the hair regions of pectin chains in the form of neutral sugars (19, 20, 42). Some arabinogalactans form compounds with proteins called arabinogalactan proteins (20). Due to the substrate specificity and regiospecificity of each hemicellulase, the destruction of a particular hemicellulose (Table 1) is best performed by a mixture of enzymes (18).

Lignin is a phenolic macromolecule consisting of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S)-type methoxylated phenylpropane units connected by different types of carbon–carbon and ether linkages, which is found in the cell walls of vascular plants (21, 43). Several enzymes are known to attack and degrade lignin, such as laccase (EC 1.10.3.2), lignin peroxidase (LiP, EC 1.11.1.14), manganese peroxidase (MnP, EC 1.11.1.13), and versatile peroxidase (VP, EC 1.11.1.16)). Laccase and peroxidases catalyze depolymerization of the lignin macromolecules with formation of phenolic hydroxyl groups, oxidation of phenolic groups to phenoxy radicals and subsequent cross-linking of lignin (17, 43).

In nature, phenol-oxidizing enzymes are produced by basidiomycetes such as white-rot fungi, but also brown-rot fungi. White-rot fungal enzymes are capable of destroying not only lignin, but also all the main components of lignocellulose, including cellulose and hemicellulose (17, 21, 43, 44). The enzymes expressed by brown-rot fungi are able to cause lignin oxidation, depolymerization, demethylation of lignin methoxy groups, and the removal of cellulose and hemicelluloses from plant cell walls (17, 43).

The technology of enzyme-assisted delignification can be used to process lignocellulosic materials into medium-density fibre board and particle board, aromatic value-added chemicals, biofuels, and in paper pulp manufacture (17, 21, 43). However, is it appropriate to use phenol-oxidizing enzymes to destroy the cell walls of oilseeds to increase oil yield? Lignin is a main component of the seed coat. The content of lignin and cellulose carbohydrates in the seed kernels is much lower than in the husks. The compositional analysis of 20 hemp seed varieties and lines revealed that the presence of these compounds in the hemp seed hulls ranged from mass fraction of 16.0 to 19.5 % for lignin and from 22.0 to 36.7 % for cellulose, while both of these components were absent from the hemp seed kernel (22). The lignin mass fraction in rice bran was in the range of 7.7–24.8 %, in the seed coats of rapeseed, sunflower and cucurbit seeds was from 4.8 to 27.1 %, while the mass fraction of lignin in the oilseed kernels varied from 0.5–0.6 % for rapeseed and sunflower seeds and to 2.0–2.9 % for cucurbit seeds (23, 45-57). It is important to consider that enzymes catalyzing the oxidation of lignin can also modify the oil, which is highly undesirable. It was found that MnP and VP are able to cause the oxidation of unsaturated fatty acids by using Mn³⁺ as a mediator with the formation of peroxide radicals that will oxidize non-phenolic β-O-4-linked lignin (17, 21). The presence of peroxide radicals of fatty acids in the oil causes an increase in its peroxide value, which is a marker of low oil quality.

The polysaccharide analysis of the hemp seed kernel detected unesterified and partially esterified homogalacturonan, and also low amounts of arabinogalactan proteins, rhamnogalacturonan I, callose, polysaccharides containing α-1,5-l-arabinan (22). Among the non-starch polysaccharides of soybeans 8 % cellulose and 17 % pectins were detected, which are cell wall constituents. The most abundant pectins were rhamnogalacturonans, arabinogalactan I and xylogalacturonan (24). Microscopic analysis of stained samples and fluorescence microscopy revealed the microstructure of oilseeds and oil-containing plant tissues (Table 1). Pectin-rich regions were detected by fluorescence staining with coriphosphine O, lignified cell walls were stained with Alcian blue, and the turquoise-blue colour represents the autofluorescence of protein bodies within the storage cotyledons.

The hemicellulose composition of plant cells is very complex. The monoclonal antibody analysis of hemicellulose composition of soybean seeds revealed galacturonan, homogalacturonan, xylogalacturonan, xyloglucan, mannan, galactan, arabinan and rhamnogalacturonan (25). The total polysaccharide composition of rapeseed includes 3.5 % arabinogalactan, 6.9 % arabinan, 15.5 % amyloid, 24.1 % cellulose and 50.0 % pectin, while the secondary cell walls of rapeseeds contain 39.0 % pectins, 29.0 % hemicelluloses, 22.0 % cellulose, 8.0 % of arabinogalactans and they make 20.0 to 28.0 % of the total seed (18, 37). Rice bran dietary fibre contains approx. 90 % insoluble dietary fibre represented by cellulose, hemicellulose and arabinoxylans, and 10 % soluble dietary fibre, such as pectin and β-glucan. Feruloyl polysaccharide in the rice bran is represented by oryzanol, which is a mixture of ferulic acid esters of sterol and triterpene alcohols (23, 39). The polysaccharide fraction of defatted Moringa oleifera seed flour consists of arabinogalactan, xylan-type polysaccharides and cellulose (41).

Despite the differences in the chemical composition of various types of oilseeds obtained in different studies, it can be concluded that the use of delignifying enzymes to destroy the cell walls of seeds in order to improve oil production may be inappropriate, because lignin is more abundant in the seed husks than in the oil-containing kernels, and the oxidases themselves pose risks to the obtained oil quality. Much more promising is the application of hydrolytic enzymes for cellulose and hemicellulose hydrolysis. The following enzymes have been used for this purpose (Table 1): exocellobiohydrolase (EC 3.2.1.91), endocellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), α-l-arabinofuranosidase (EC 3.2.1.55), α-l-arabinanase (EC 3.2.1.99), acetylxylan esterase (EC 3.1.1.72) and feruloyl esterase (EC 3.1.1.73). It is also possible to divide into two separate subgroups xyloglucan-degrading enzymes, such as xyloglucan-specific endo-β-1,4-glucanase (EC 3.2.1.151), xyloglucan-specific exo-β-1,4-glucanase (EC 3.2.1.155), oligoxyloglucan β-glycosidase (EC 3.2.1.120), and arabinogalactan-degrading enzymes, represented by galactan-1,3-β-galactosidase (EC 3.2.1.145), galactan endo-β-1,3-galactanase (EC 3.2.1.181), galactan endo-1,6-β-galactosidase (EC 3.2.1.164) and arabinogalactan endo-β-1,4-galactanase (EC 3.2.1.89). Because oilseed kernels also contain many pectin substances, including homogalacturonans and rhamnogalacturonans, the following enzymes have been used for their degradation: endopolymethylgalacturonate lyase (EC 4.2.2.10), exopolymethylgalacturonate lyase (EC 4.2.2.27), pectinesterase (EC 3.1.1.11), and rhamnogalacturonan-degrading enzymes, such as rhamnogalacturonan hydrolase (EC 3.2.1.171), rhamnogalacturonan galacturonohydrolase (EC 3.2.1.173), rhamnogalacturonan rhamnohydrolase (EC 3.2.1.174) and rhamnogalacturonan endolyase (EC 4.2.2.23) (Table 1).

The cotyledon is the main tissue in oilseeds, where proteins and lipids are accumulated and stored in the form of protein and lipid bodies. Lipid bodies in seed cells are woven into a cytoplasmic membrane consisting of proteins and also externally protected by a stable pectin and lignocellulosic matrix of seed cell walls (12, 13). The epidermal cells of the Moringa oleifera seed cotyledon (image j in Table 1), as well as the rapeseed cotyledon cells (image d in Table 1), are occupied by lipid bodies, whereas protein bodies are seldom present and starch grains are totally absent (36, 40). Subepidermal cells of the cotyledon contain lipid bodies surrounded by the protein bodies and they fill most of the remaining space in all healthy cells (40). Most of the rice bran lipids are in a form of lipid bodies in the aleurone layer (23). Based on the structure of lipid bodies to intensify their destruction and improve oil extraction from oilseeds, it is advisable to use proteases that destroy not only proteins of the oleosome membranes, but also glycoproteins in the cell walls and cytoplasmic membranes, including arabinogalactan proteins.

Among proteolytic enzymes, proteases of animal, plant, fungal and bacterial origin with broad specificity deserve attention (Table 2 (58-96)). Nowadays, various proteases are used by researchers to improve the production of protein products from natural sources, including oilseeds. It was proposed to prepare soy protein hydrolysates selectively enriched with the soybean protein glycinin and β-conglycinin by hydrolysis with pepsin and papain, respectively (97). Rapeseed protein isolate was hydrolyzed with Alcalase, Proteinase K, pepsin+pancreatin (P+P), thermolysin and Flavourzyme under different conditions (98). Porcine pepsin and pancreatin treatment were used to obtain sunflower protein hydrolysates (99). Bromelain and papain, which are used for tenderizing squid (Loligo vulgaris) muscle, are also potentially useful proteases (58). The hullless pumpkin oil cake protein isolate was hydrolysed by pepsin (100). In addition, the effectiveness of pepsin for the destruction of oleosomes has also been proven. Gastric digestion of a walnut oil bodies by pepsin was investigated in vitro. The results of the experiment revealed that such kind of treatment causes destabilization and coalescence of the oleosomes (101).

Despite the beneficial effect of proteases on the oil yield, it is important to remember that some preparations such as pancreatin contain not only various proteases and amylases, but also lipases, which catalyze the hydrolysis of triacylglycerols (59). Such an effect is positive for the production of protein isolates or protein hydrolysates since it allows to increase product purity, but the presence of lipases for oil production is highly undesirable because it promotes the destruction of lipids and the release of free fatty acids (FFA). An increased amount of FFA in oil is a marker of reduced quality. Therefore, during the enzymatic treatment of oilseeds and oil-containing plant materials, it would be appropriate to use a combination of pepsin with another pancreatic enzyme like trypsin (EC 3.4.21.4) or elastase (EC 3.4.21.36) instead of pancreatin.

The destruction of the phospholipid component of oleosome membranes is facilitated by the use of phospholipases and phosphatases. Phosphatases and phospholipases are very common in nature and regulate metabolic processes in animal, plant and microorganism cells. Phospholipases hydrolyze phospholipids and release lysophospholipids, free fatty acids, diacylglycerols, choline phosphate and phosphatidates, while phosphatases catalyze the hydrolysis of phosphomonoesters (59, 74-96, 102, 103). Traditionally, phospholipases are used in the industry for degumming of vegetable oils, as a replacement for emulsifiers in bread making to increase the dough stability, in dairy to increase cheese yield by hydrolysis of milk phospholipids, and for the modification of egg yolk in mayonnaise preparation (74). Most phospholipases catalyze the hydrolysis of phosphatidyl choline (Table 2), which, as mentioned above, predominates in the phospholipid layer of oleosomes. Phospholipids are also important components of all cell membranes (14, 15, 74-85). It was found that the destruction of phospholipids by phosphatases detected in oilseeds causes the destruction of cell membranes. Protein bodies of the Moringa oleifera seed cells contain several hydrolytic enzymes and phosphatases and their release into the cytosol causes localized cellular autolysis and membrane deterioration (40). Therefore, the prospect of possible use of phospholipases and phosphatases for the destruction of seed cell membranes and oil body membranes could be a strategy to increase the oil yield during pressing.

At the same time, this enzymatic treatment should be used with caution. Authors who studied the effect of phospholipases in the degumming of vegetable oils on their quality reported an increase in the peroxide value of sunflower oil after treatment with phospholipase A₁, and a decrease in oxidative stability of rapeseed oil after degumming with phospholipase C and soybean oil with each of phospholipases A₁ and C (80-82). Such changes can probably be explained by the fact that the destroyed phospholipids could play the role of natural antioxidants in oils (81, 82).

Given the multicomponent composition of oilseed cells, it can be deduced that for a maximum oil release it is important to destruct the polysaccharides which are cell wall components together with proteins and possibly also phospholipids that are components of the cytoplasmic membrane and oleosome membranes. For such a strategy a mixture of enzymes with different activities is suggested. For example, enzymes used to hydrolize plant protein isolates contain a combination of proteases (brand names) like Alcalase (Bacillus licheniformis, P4860), Neutrase (Bacillus amyloliquefaciens, P1236) and Flavourzyme (Aspergillus oryzae, P6110) (104). For the production of protein isolates from pumpkin seeds, a mixture of pectinase and cellulase in a ratio of 1:1 was employed (105) and for the extraction of polysaccharides from bamboo shoots or ginger stems, an enzyme mixture of cellulase, papain and pectinase in a ratio of 1:1:1 worked well (106, 107).

The use of enzymes with different activities has also been investigated for the extraction of vegetable oils from seeds, e.g. a mixture of proteases and cellulases in different ratios and under different conditions in order to increase the yield of pressed oil from pumpkin seed and rapeseed (108-110). Many researchers have treated various oilseeds with mixtures consisting of enzymes with proteolytic, cellulolytic and pectolytic activity (6, 9, 13, 111, 112). Some studies have also used the treatment of oil material with separate enzyme preparations, such as Ronozyme VP (endoglucanase, hemicellulase and pectinase) from DSM Nutritional Products (Basel, Switzerland), Protex 7L (protease) and Multifect CX 13L (cellulase, β-glucanase, hemicellulase and arabinoxylanase) from Genencor (Rochester, NY, USA), Viscozyme L (cellulase, β-glucanase, hemicellulase, arabanase and xylanase) from Novozymes (Bagsvaerd, Denmark), Natuzyme (cellulase, α-amylase, pectinase, xylanase and phytase) from Bioproton Pty Ltd (Acacia Ridge, Australia), and Kemzyme (protease, cellulase, β-glucanase, α-amylase, hemicellulase and xylanase) from Kemin Europa N.V. (Herentals, Belgium) (11, 25, 113, 114). In vitro non-starch polysaccharide analyses of solubilized soybean meal cell walls after treatment with the multienzyme product Ronozyme VP (Aspergillus aculeatus) revealed a statistically significant reduction in insoluble sugar residues for (in %): rhamnose 35, arabinose 36, galactose 36 and glucose 39. The solubilisation of xylose, mannose and uronic acid was about 18, 14 and 22 % respectively. The specific degradation of pectin homogalacturonan epitopes, β-1,4-mannan, xyloglucan, galactan and arabinan of soybean meal cell walls was detected with monoclonal antibodies (25). The treatment of Moringa oleifera seed with Protex 7L increased the oil yield by 69.4 % compared to all other enzymes, which can be explained by its proteolytic activity and, consequently, by hydrolysis of proteins that are part of lipid spherosome membranes, unlike other enzymes, which were characterized by more cellulolytic and pectolytic activity (114).

The technology of oilseed preparation for oil extraction by pressing includes operations of seed reception, cleaning and weighing, dehulling, flaking and cooking (142, 143). All these operations affect or are affected by enzymatic pretreatment. To prevent spoilage of seeds during long-term storage in the silos, as well as to facilitate their further technological processing and to obtain a high-quality product, seeds should be cleaned (143). Dehulling is the next operation after cleaning; this process usually consists of two stages: opening the seed coats by cracking and separating the hulls from the kernels by screening and aspiration. The presence of a certain amount of hulls in the oleaginous material is necessary to ensure its desired structure during pressing. However, the hull material is also a substrate for many of the reported hydrolases and would be a competitive substrate to the oilseed cells, thus reducing the enzymatic action on the oilseed cells. An approximate hull content of 8.0 % is recommended for sunflower kernels that undergo flaking and further processing. However, for some types of oilseeds that are very small in size and have the seed hulls firmly stuck to the kernels, the process of dehulling is impractical. For example, flaxseed and rapeseed are flaked immediately after cleaning (142, 143). Small particles of oil-rich material will stick together and decrease the porosity. Therefore, the particle size after grinding should be optimal (142). The analysis of works devoted to the application of enzymatic treatment of oil-containing material before oil extraction showed that the process is effective if the particle size of the oilseeds is in the range of 0.5–1.0 mm (6, 11, 111-113, 117-119). At the same time, according to the recommendations of the traditional technology of oil extraction from oleaginous material by pressing, the particle size should be 0.4–1.0 mm for sunflower seeds, approx. 1.0 mm for flax- and cottonseeds, and 0.25–0.30 mm for soybean seeds (142, 143). Therefore, the particle size of the seeds in the range of 0.3–1.0 mm can be considered optimal for enzyme-assisted mechanical pressing, smaller diameters result in a higher specific surface area and provide a better accessibility of the plant structures by enzymes.

Cooking is a key step in preparing oilseeds for oil extraction by pressing. This process involves the simultaneous or sequential treatment of oleaginous material with water and heat in order to change its colloid-chemical and physico-mechanical properties, and to weaken the cell walls. Cooking is also a necessary operation for inactivating enzymatic activity, preventing the hydrolysis of triacylglycerols and development of mould and bacteria, as well as for binding anti-food substances, such as gossypol in cottonseeds (142-145).

Oilseeds contain their own set of enzymes that catalyze complex biochemical processes during storage and processing, which will affect the quality of the obtained oil. Lipases from oilseeds cause hydrolysis of triacylglycerols, accompanied by an increase in free fatty acids in the oil (142-145). In rice bran, lipase is so active that in the first hours of storage the acidity of crude oil increases at a rate of 1 % per h, 5–7 % per day, and can finally reach up to 50 % of free fatty acids (143, 146). Flaxseeds, in addition to lipase, also contain the glucoside linamarin and the enzyme linase, which catalyzes the cleavage of its glucosidic bonds with the release of hydrocyanic acid. Press cake containing hydrocyanic acid can be dangerous to livestock during feeding (142, 143). The effect of high humidity and temperature of 40–60 °C in the process of cooking will significantly increase the activity of these enzymes, while their inactivation requires short-term (30–40 s) heating of wet oilseeds at 80–90 °C. In industry, inactivation is carried out at the first stage of cooking in a separate screw steamer, or in the roaster, where all subsequent stages of cooking take place (142, 144, 145). It is recommended to sterilize rice bran immediately upon arrival at the plant by heating at 90–100 °C and drying to stop lipase activity, or to stabilize it by heating at 125–135 °C for 1–3 s at 11–15 % moisture or by wet extrusion at 120 °C and 10 % added water as steam (143, 146).

During cooking, the hydrophilic gel particles increase significantly in volume, and as a result, the volume of all oil-filled cavities decreases, and the oil is pushed to the surface of the particles under the action of swelling pressure (142, 145).

At the moistening stage during cooking in the industry, the oil-containing material is usually adjusted to the humidity values of 8.0–9.0 % for sunflower seeds and flaxseeds and 11.5–17.5 % for cottonseeds (142, 144, 145). However, for enzymatic pretreatment of oilseeds, the optimal amount of added water should be in the range of 20–50 % (3, 4, 9-11, 108-110, 119, 131, 132). A water content below 20 % may not be enough to fully carry out the enzymatic hydrolysis and to evenly distribute the dissolved enzyme between the particles of the material, while increasing the water level above 50 % will cause an increase of time and energy during drying and roasting. Under industrial conditions, the amount of water added to the oleaginous material during cooking is usually much smaller than the theoretically possible amount that can be absorbed and bound. For example, the maximum swelling of ground sunflower seed kernels is achieved at 35 % of the added water, which will be completely absorbed and bound. When the oil-containing material is overwetted, the processes of oil displacement by water become much more intense, due to swelling, which allows oil to be easily separated from the material even with very little external pressure (142). Already in 1929–1934, a method was invented to prepare oilseeds for oil extraction, which involves treating ground seeds with water or hot steam for 2–7 min at a temperature of 20–80 °C with bringing the oil-containing material to a humidity of 12–20 %. The method allowed to extract 60–70 % of high-quality oil from seeds; however, its disadvantages were the difficulty of introduction into continuous process and the high degree of denaturation of protein compounds in the material (142, 147). Although this method has not become widespread in industry and has been quickly replaced by other technologies currently used in oil production, its basic idea allows us to make the assumption that adding more water (20–50 %) to the oleaginous material during enzymatic pretreatment may be acceptable for cooking.

The wet material is excessively ductile, which makes it impossible to use for pressing. To remove excess moisture from the oil-containing material and provide a more rigid structure necessary for its further processing, it is first dried and then roasted by gradually raising the temperature. Under the action of heat, the moisture begins to evaporate from the lower layers of the processed material and passes successively through the middle and upper layers, resulting in a process of self-evaporation of oleaginous material (142, 144, 145). Heating to 50–60 °C causes a sharp decrease in its oil viscosity, while with a further increase in temperature, the viscosity changes are less significant (142). At the same time, a temperature range from 40 to 55 °C is the optimal for enzymatic pretreatment of oilseeds, at lower temperatures the process will not be efficient enough, and above 60 °C denaturation of the enzyme protein part and its inactivation will occur (3,4,6,9,10,13,108-114,117,118,130131,133,134).

During roasting, the temperature gradually rises from 80 to 105 °C, which causes denaturation of proteins, aggregation of particles, and the material becomes more rigid. Carrying out the process at temperatures above 105 °C is undesirable, as it will intensify the oxidation processes in the obtained oil. The final moisture content of the oil material after roasting has to be 5.0–6.0 % for sunflower seeds, 4.5–5.0 % for flaxseeds and 4.6–6.0 % for cottonseeds (142, 144, 145).

A possible application of the enzyme-assisted mechanical pressing in industry is shown in Fig. 2, where the enzymatic treatment of oil-containing material is carried out in the mixer-expositor (3) during cooking, i.e. after the stage of seed enzyme inactivation in the screw steamer (2), and before the stage of roasting material in the roaster (6). The enzyme solution with the required pH=4.5–5.5 is prepared in a tank (5) and fed to the mixer-expositor (3). The enzymes added to the oleaginous material are inactivated in the roaster (6).

In the analysis given in the previous section, it was found that 1.5–2.0 h is the optimal duration of the oil-containing material incubation with the enzymes before pressing, excluding time for inactivation and roasting, while the duration of traditional cooking in industry, including inactivation, humidification and roasting, usually is 40–45 min (108-110, 113, 114, 133, 141, 142). A twofold increase of the process duration is a significant disadvantage of this technology and might be difficult to implement in continuous industrial process. However, enzyme-assisted pressing can be effectively used in smaller enterprises with a non-continuous, periodic production.

Nowadays, enzymatic pretreatment of oil-containing plant materials and oilseeds is investigated mainly at the laboratory scale and only in a few pilot studies. To transfer this technology to the industry, it has to increase the yield of oil extraction significantly, but not to interfere with the established processes. A key factor is the treatment time, which has a strong influence on technical applicability, commercial feasibility and product quality. Therefore, it is important to optimize the applied enzyme cocktails and the key parameters of the enzymatic pretreatment to keep the process time short, the particle size big enough to maintain porosity for pressing, and the moisture content as low as possible. Other factors such as temperature and pH during enzymatic pretreatment are easier to adapt to increase enzyme performance. The biggest factor is, however, an optimal combination of enzymes for a given substrate that attacks not only the cell wall, but also the membrane oleosome simultaneously and combines different strategies to weaken the cell microstructures for efficient oil extraction. The cell structure is a very complex and multicomponent system and varies with the type of seed. In order to make efficient use of enzymatic pretreatment, its chemical composition has to be investigated and the enzymes have to be selected accordingly. A beneficial outcome of the enzymatic pretreatment might be a reduced pressure during pressing, which reduces the energy demand and costs. The combination of enzymatic pretreatment with mechanical extraction by pressing has the potential to be more eco-friendly than solvent oil extraction methods. Due to the different properties and composition of various oilseeds, each of them needs an individual optimization for pretreatment.

The increased hydrolysis of plant biopolymers can also intensify the recovery of other valuable components, such as carbohydrates, protein isolates, or protein hydrolysates from the plant biomass. In combination with the improved oil recovery, this can ensure waste-free production and promote the efficient use of renewable materials.