With the rising importance of enzyme-assisted extraction of essential oil from plant and microbial sources, researchers are now interested in studying the effect of combination of enzyme-assisted extraction and other non-conventional extraction techniques on the extraction of essential oil. Non-conventional techniques such as three-phase partitioning, microwave-assisted extraction, ultrasound-assisted extraction, and supercritical fluid extraction (Gupta et al. 2012). The combination of enzyme-assisted extraction and these techniques can be considered economical as well as an efﬁcient method to extract essential oil.
Enzyme-Assisted Three-Phase Partitioning (EATPP)
Three-phase partitioning (TPP) is a new technique used to separate proteins by precipitating them using t-butanol and ammonium sulfate. The method usually used for separation of proteins is now being studied for its use in extraction of bioactives such as oil and carbohydrates primarily from plant sources. In TPP, the proteins are separated from aqueous phase by adding t-butanol and ammonium sulfate which forms two immiscible liquid phases to precipitate the proteins at the interface of two layers (Gaur et al. 2007).
TPP offers more advantages over conventional protein extraction methods, which includes the use of mild operational conditions and structural stability of proteins in their native form. TPP can further be used to scale-down or scale-up of processes. Moreover, it can be employed directly on crude plant materials to reduce the process cost. The use of inexpensive chemicals such as t-butanol and ammonium sulfate also makes the process commercially valuables (Rachana and Lyju Jose 2014).
Enzyme-assisted three-phase partitioning (EATPP) is an advanced technique which is a combination of enzyme-assisted extraction and TPP. The plant material is pretreated with enzyme preparations followed by regular TPP process. Sharma et al. (2002) performed TPP for the extraction of oil from soybean and obtained a yield of 82% within 1 hour. Similar experiments were carried out using EATPP with the help of Protizyme (a protease) to extract oil from soybean and obtained a yield of 98% (Gaur et al. 2007). This shows the better efﬁciency of EATPP over the TPP. Kurmudle et al. (2011) carried out EATPP for the extraction of turmeric oleoresin by pretreating the turmeric slurry with a commercial preparation of enzymes such as a-amylase and/or glucoamylase. Oleoresins were extracted in less time as compared to conventional acetone extraction. Harde and Singhal (2012) also used this method for the extraction of forskolin (diterpene) from Coleus for- skohlii roots. The extraction was found to be increased from 30.83% by TPP to 83.85% by EATPP.
Microwave-Assisted Enzymatic Extraction
Microwaves are electromagnetic radiations with their frequencies ranging from 300 MHz to 300 GHz. These are non-ionizing radiations and can cause molecular motion on contact with matter without changing the molecular structure. Also, they can heat the target material, and the amount of heat generated greatly depends on their frequencies and on the applied power. Microwaves can be used in the extraction of bioactive compounds, and the yield of extraction depends on several factors such as the power of microwaves, time for which the material is exposed to microwaves, size of sample, extractant (solvent), and temperature. The choice of solvent affects the extraction process due to the solubility of compound in solvent and the ability of a solvent (extractant) to absorb microwave energy. Higher absorption might generate high heat leading to effective extraction.
Microwave-assisted extraction (MAE) has been studied extensively by various researchers for the extraction of different bioactives (Pan et al. 2003; Lianfu and Zelong 2008; Chen et al. 2007). Moreover, microwave-assisted enzymatic extraction (MAEE) was used by Yang et al. (2010) for the extraction of corilagin and geraniin from Geranium sibiricum Linne. The increased yield of bioactives with good potential for natural antioxidant was found in the extract. Similar technique was explored by Zhang et al. (2013) to enhance the extraction of polyphenols from the waste peanut shells. The yield of polyphenol obtained was higher than other methods such as heat-refluxing extraction, ultrasonic-assisted extraction, and enzyme-assisted extraction. Cellulase is a common enzyme used in MAEE where it makes the process efﬁcient and environmentally friendly. Recently, the method has also been used for the extraction of polysaccharides from the fruits of Schisandra chinensis Baill (Cheng et al. 2015) where the yield obtained was higher at low temperature.
Ultrasound-Assisted Enzymatic Extraction
Ultrasonic waves are sound waves with high frequencies (20 kHz–100 MHz) and are not audible to humans. Ultrasonic waves have been used for several purposes such as cleaning, atomization, and extraction. Ultrasonic waves cause cavitation that results in disintegration of material; this property of ultrasonic waves has been utilized for extraction procedures. It displays several advantages over other extraction methods such as reduced processing time, higher extraction rate, and better extract quality (Cravotto et al. 2004).
Ultrasonic waves cause vibrations in the extractant leading to the formation of bubbles which collapse near the cells and cause a shock wave. This leads to breakage of cells and release of cell contents in the extractant. Ultrasound-assisted extraction (UAE) has already been proven to be better than other methods such as microwave-assisted extraction and simple aqueous extraction (Gu and Pan 2014). UAE technique can further be improved by combining it with enzyme-assisted extraction (EAE). Ultrasonic-assisted enzymatic extraction (UAEE) is a perfect combination of enzymolysis and ultrasonication which shows efﬁcient extraction of polysaccharides from Cucurbita moschata and arabinoxylan, a major dietary component from wheat bran (Wang et al. 2014). The method has been optimized with respect to temperature, pH, ultrasonic power, liquid-to-material ratio, enzyme dose, and time of extraction. Recently, Pu et al. (2015) have optimized the UAEE method for the extraction of polysaccharides from Atratylodes macrocephala using response surface methodology and have recommended this method as appropriate and efﬁcient.
Enzyme-Assisted Supercritical Fluid Extraction
Supercritical fluid extraction has been widely used for the extraction of alkaloids, flavonoid (Giannuzzo et al. 2003), catechin, and epicatechin (Ashraf-Khorassani and Taylor 2004) from different sources. This is a relatively new technique in the ﬁeld of extraction. In recent times, enzyme-assisted supercritical fluid extraction (EASFE) has started gaining attention. The source raw materials are pretreated enzymatically, and the bioactives of interest can be extracted using supercritical fluid extraction technique. Mushtaq et al. (2015) have extensively studied this method for the ex- traction of antioxidant phenolics from pomegranate peels. EASFE could produce crude extract of double recovery with increased level of phenolic constituents, improved radical scavenging capacity, trolox equivalent antioxidant capacity, and inhibition of linoleic acid peroxidation. Further, Dutta and Bhattacharjee (2015) have used a-amylase in the process of EASFE to extract black paper oleoresin. This method not only enhanced the yield of the oleoresin but also improved the phyto- chemical properties of oleoresins. The extraction was studied comprehensively by using batch and continuous mode where the most signiﬁcant results of yield were obtained with batch mode of operation.
Large-Scale Enzymatic Processes
As evident from foregoing review, several enzymes are being used for extraction of biomolecules and now traded as commodity products globally. Although the cost of enzymes for use at the research scale is often very high, the increased production and multiple use of enzymes reduce the cost dramatically. Enzymes are currently involved in industrial processes with annual turnovers totaling many billions of dollars.
Cell wall-degrading enzymes can be used to extract oil by solubilizing the structural cell wall components of the oilseed. This concept has already been commercialized for the production of olive oil and has also been investigated for other oil-bearing materials (Christensen 1989). The enzyme cocktail works synergistically to give better results than individual enzymes. Many enzymes have been commercialized for the industrial enzymatic extraction processes and have been reported well in the literature as explained in different sections of this chapter. Enzymatic treatment also destabilizes the lipophilic extractives in the ﬁltrates and facilitates their attachment to thermomechanical pulping ﬁbers. The enzymes are also used in the preparation of easily biodegradable cardboard (Buchert et al. 1998), manufacturing of soft paper including paper towels and sanitary paper (Salonen 1990; Hsu and Lakhani 2002), and removal of adhered paper (Sharyo et al. 2002). In recent years, extraction of olive oil has attracted the interest of international market because of its numerous health claims. To produce high-quality olive oil, freshly picked, clean, and slightly immature fruits are used under cold pressing conditions (Galante et al. 1998; De Faveri et al. 2008). Although high yields are obtained with fully ripened fruit, when processed at higher than ambient temperatures, these process conditions result in poor oil quality with high acidity, ran- cidity, and poor aroma (Galante et al. 1998). Hence, an improved method for the extraction of high-quality olive oil was needed to meet the growing consumer demand. The commercial enzyme preparation, Olivex (a pectinase preparation with cellulase and hemicellulase from Aspergillus aculeatus), was the ﬁrst enzyme mixture being used to improve the extraction of olive oil (Fantozzi et al. 1977). Furthermore, the use of macerating enzymes increased the antioxidants in extravirgin olive oil and reduced the induction of rancidity (Galante et al. 1998). The main advantages of using macerating enzymes during olive oil extraction are as follows: (i) increased extraction (up to 2 kg oil per 100 kg olives) under cold processing conditions; (ii) better centrifugal fractionation of the oily must; (iii) oil with high levels of antioxidants and vitamin E; (iv) slow induction of rancidity; overall improvement in plant efﬁciency; and (vi) low oil content in the wastewater (Galante et al. 1998). Likewise, the macerating enzymes could play a prominent role in the extraction of oils from other agricultural oilseed crops.
In wine production, enzymes such as pectinases, glucanases, and hemicellulases play an important role by improving color extraction, skin maceration, must clar- iﬁcation, ﬁltration, and ﬁnally the wine quality and stability (Singh et al. 2007; Galante et al. 1998). A number of commercial enzyme preparations are now available for use by the wine industry. The main beneﬁts of using these enzymes during wine making include better maceration, improved color extraction, easy clariﬁcation, easy ﬁltration, improved wine quality, and improved stability (Galante et al. 1998).
Cellulases have a wide range of potential applications in food biotechnology. The production of fruit and vegetable juices requires improved methods for extraction, clariﬁcation, and stabilization. Cellulases also have an important application as a part of macerating enzymes complex (cellulases, xylanases, and pectinases) used for the extraction and clariﬁcation of fruit and vegetable juices to increase the yield of juices (Minussi et al. 2002; De Carvalho et al. 2008). Enzyme mixtures containing pectinases, cellulases, and hemicellulases are also used for the improved extraction of olive oil. Thus, the macerating enzymes, composed of mainly cellulase and pectinase, play a key role in food biotechnology, and their demand will likely to increase for the extraction of juice from a wide range of fruits and vegetables (Dourado et al. 2002).
Challenges and Future Perspectives
Extensive research has been carried out in large-scale application of enzyme- assisted extraction of biomolecules. However, there are various challenges associated with cost-effective applications in current commercial processes. The possible solutions for further commercialization of enzymes in extraction industry include the following: (a) reduction in the cost of enzyme production, (b) improvement in the performance of enzymes by using protein engineering and genetic engineering, and (c) repeated use of enzymes with the help of improved enzyme immobilization techniques. In the past decade, commercial enzyme companies have made signiﬁcant progress in producing new generations of enzymes with higher speciﬁc activities and lower cost using different biotechnology and process engineering approaches. However, a techno-economic analysis suggests further progress be made for better commercial applications. Another novel approach is expressing enzymes in plants that could be extracted and used after pretreating the extracted biomass (Egelkrout et al. 2012). Enzymes could also be produced directly in bioreﬁneries rather than producing them in a centralized location. Producing enzymes on-site at bioreﬁneries would eliminate the need for concentration, storage, and shipping and could reduce the production costs by using pretreated substrates already available at the bioreﬁnery (Culbertson et al. 2013).