Enzyme-Assisted Extraction of Bioactives

Chapter 8
Enzyme-Assisted Extraction of Bioactives
Sandesh J. Marathe, Swati B. Jadhav, Sandip B. Bankar

and Rekha S. Singhal
1 Introduction
Among the vast array of biomolecules present in the living organisms, some of
them are essential for survival and are called primary metabolites. However, other
biomolecules called as secondary metabolites are also naturally produced. These are
extra nutritional in nature and may have a beneficial or adverse effect on living
organisms (Amsath 2013). Secondary metabolites have limited distribution in
nature and present only in the specific group of organisms. Biomolecules from plant
and microorganisms have been used for centuries, and their demands have
increased in food, medicinal, and chemical industries due to their unique biological
activities. Bioactive compounds are constituents other than nutrients that generally
occur in small quantities of foods, and whose intake has been associated with
protective effects against adverse health or physiological disorders, for instance,
cardiovascular, diabetes, or cancer. These bioactives have a diverse range of
chemical structures varying from phenolic compounds to phytoestrogens to car-
otenoids to terpenoids to organosulphur compounds among many others. The
efficacy of bioactives has been established from epidemiological studies as well as
in vitro and in vivo studies in both animals and humans. The discovery and efficacy
of bioactives are now the basis of a billion dollar nutraceutical industry globally.
One of the main challenges is to extract these biomolecules from their respective
natural sources. Different techniques have been reported on these aspects in the
literature, each having their own pros and cons. The choice of technique mainly
S.J. Marathe S.B. Jadhav R.S. Singhal (&)

Food Engineering and Technology Department, Institute of Chemical Technology,
Matunga, Mumbai 400 019, India
e-mail: rs.singhal@ictmunbai.edu.in; rsinghal7@rediffmail.com
S.B. Bankar
Department of Chemical Engineering, College of Engineering, Bharati Vidyapeeth
University, Pune-Satara Road, Pune, India
© Springer International Publishing AG 2017 171

M. Puri (ed.), Food Bioactives, DOI 10.1007/978-3-319-51639-4_8
172 S.J. Marathe et al.
depends on the type of raw material, environmental concerns, process conditions,

and future applications of the bioactives.
Enzyme-assisted extraction offers safe, green, and novel approach for the
extraction of bioactives. Several researchers have reported this method as a best
choice for extraction of bioactives from various sources. Enzyme-assisted extrac-
tion has been reported for the extraction of lipids (Jin et al. 2012), oils (Huo et al.
2015), and various other bioactives from plants (Puri et al. 2012).
This chapter covers a brief reference to conventional extraction technique and
the importance of enzyme-assisted extraction of biomolecules. It also covers the
mechanism of action and various hyphenated technologies using enzymatic
extraction of different types of bioactives.
2 Conventional Extraction Techniques
Over the years, different extraction techniques have been reported and used for the
extraction and purification of the biomolecules from the natural resources. The low
content of active molecules in the source material and the complexity of raw
material makes it necessary to find alternative methods of effective extraction. It is
worthwhile to understand the conventional methods of extraction before discussing
enzyme-aided extractions.
2.1 Solvent Extraction Methods
Solvent extraction is the oldest and traditional method of extraction which mainly
depends on factors such as nature of the solvent, energy input, and agitation to
improve the chemical solubility and efficiency of mass transfer (Awika et al. 2003).
The selection of solvent for the extraction depends on the raw material to be used
and the product of interest. Lipophilic compounds can be well extracted using
nonpolar organic solvents such as hexane or dichloromethane, whereas hydrophilic
compounds can be extracted using polar solvents such as acetone, methanol, or
ethanol. Mixture of acetone and water has been used for the extraction of antiox-
idant (Awika et al. 2003). The recovery of polar compound such as lignin glyco-
sides can be further enhanced by adding polar solvents such as water to the sample
(Cacace and Mazza 2006). The use of solvents such as dichloromethane, dichlor-
oethane, acetone, hexane, and alcohol is very common for the extraction of aroma
principles from various raw materials either in hot or in cold conditions (Ravindran
and Madhusoodanan 2002). Xu and Chang (2007) have demonstrated the influence
of solvent type on the rate and amount of extraction of polyphenols. Polyphenols
are commonly extracted using solvents such as methanol, ethanol, acetone, ethyl
acetate, and their combinations with different proportions of water. The lower
molecular weight polyphenols can be extracted effectively using methanol, while
8 Enzyme-Assisted Extraction of Bioactives 173
higher molecular weight polyphenols have shown preference toward aqueous

acetone (Metivier et al. 1980). However, ethanol has also been considered as a good
solvent for polyphenol extraction which is safe for human consumption (Shi et al.
2005).
The addition of organic acids such as formic acid, acetic acid, citric acid, and
phosphoric acid in the solvent is also practiced to increase the yield of anthocyanins
by denaturing the cell membranes and stabilizing the anthocyanins (Nicoué et al.
2007). Further, sulfured water can also be used to reduce the use of organic solvents
as well as the cost of extraction (Cacace and Mazza 2002).
Solvent extraction has many disadvantages such as (i) consumption of large
amount of organic solvents or water during extraction step, (ii) separation of solute
that needs high energy, (iii) coextraction of impurities, and (iv) chances of degra-
dation of thermosensitive compounds such as carotenoids.
2.2 Physical Extraction Methods
Essential oils from spices such as pepper, ginger, and cardamom can be obtained
using steam distillation. Aroma-rich raw materials are subjected to steam distilla-
tion, where the volatile compounds condense and separate from water (Ravindran
and Madhusoodanan 2002). In the process of hydrodistillation, the raw material is
boiled with water. The steam carries the volatile aroma compounds and condenses
it. Distillation method is not generally useful for industrial purposes as it requires
longer time. However, supercritical fluid extraction (SCFE) is one of the recent
techniques which uses the gases above its critical temperature and pressure. In the
supercritical state, the physicochemical property of the gas is intermediate between
liquid and gas. This state of the gas can effectively extract flavors and bioactive
compounds from plant materials. The use of carbon dioxide gas is very common for
this technique. SCFE has many advantages including the absence of solvents and
high concentration of compound of interest in extract (Mukhopadhyay 2000).
However, this method adds cost in the process.
Following are the limitations of using physical and solvent methods of
extraction:
1. The necessity of pretreatment to raw material;
2. The use of chemicals and solvent. These solvents are usually not recycled and
hence increase the cost of process. The cost of process also increases by adding
removal step for hazardous waste;
3. Non-specificity of the methods;
4. Variation in the quality of product might be due to remaining unwanted
materials; and
5. Low extraction yield.
3 Enzyme-Assisted Extraction
Enzyme-assisted extraction can be considered as a boon to overcome the limitations

of the conventional methods. It is a green approach and helps to reduce the issues of
environmental concerns. The most commonly used enzymes for the extraction of
bioactives are cellulases, hemicellulases, and pectinases. The major sources of
enzymes are from bacteria and fungi but can also be obtained from animal organs
and/or vegetable/fruit extracts.
3.1 Mechanism of Action of Enzyme
The mechanism of enzymatic action is the heart of enzyme-assisted extraction

process because of its selectivity. Enzyme-aided extraction is primarily dependent
on the ability of enzymes to hydrolyze the cell wall components and to disrupt the
structural complexity of the cell wall. These processes allow the easy release of
compound of interest in the bulk solution (Gardossi et al. 2010). The mechanism by
which enzyme hydrolyzes the cell wall is by forming an enzyme–substrate binding
complex. The enzyme binds to substrate with the change in conformation of the
enzyme that enables better interaction with the substrate. These changes cause
stress and strain on the substrate which in turn promotes the hydrolytic reactions
(Sowbhagya and Chitra 2010). In the enzyme-aided extraction process, the oper-
ational conditions such as temperature of reaction, pH of system, enzyme con-
centration, particle size of substrate, and time of extraction are important. The use of
appropriate enzyme complex breaks down the bonding of unwanted material and
releases the compound of interest in the aqueous media, which in turn enhances the
quality of product. Some enzymes used for the extraction of bioactives are compiled
in Table 1.
The particle size of a raw material decides the extent of hydrolytic action of the
enzymes. It is worthwhile to understand the catalytic property, mode of action, and
optimal operational conditions of the enzymes before considering its application for
the process. The types and combination of enzymes depend on the nature of raw
material (Puri et al. 2012). For instance, if a source raw material is plant, then one
needs to know the complex arrangement of polysaccharides in the cell wall.
Primary cell walls of a variety of higher plants have common structure, which
consists mainly of cellulose fibers with hemicelluloses attached to it (Talmadge
et al. 1973). These fibers are buried in a matrix made up of pectic substances which
all together are linked to a structural protein. This basic understanding of cell wall
structure gives a clue that the enzyme preparation that can be used for the
hydrolysis of cell wall must contain a mixture of cellulases, hemicellulases,
pectinases, and proteases (Christensen 1989). In the mixture of enzymes, the role of
carbohydrases (pectinase, cellulase, and hemicellulase) is different from those of
proteolytic enzymes. For example, both types of enzymes play their individual roles
Table 1 Different enzymes used in the process of enzyme-assisted extraction of bioactives from
variety of sources
Enzyme used Bioactive Source material Conditions used Reference
extracted
Cellulase Polysaccharides Garlic Temperature Pan and Wu
45 °C, pH 5.0, (2014)
time 80 min
a-Amylase Oleoresin Turmeric – Kurmudle et al.
and (2013)
glucoamylase
Cellulase, Polysaccharides Alfalfa Temperature Wang et al. (2013)
papain, and 52.7 °C, pH
pectinase 3.87, time
2.73 h
Cellulase, Seed oil Pumpkin Temperature Jiao et al. (2014)
pectinase, and 44 °C, time
protease 66 min
Alginate lyase Fucoxanthin Undaria Temperature Billakanti et al.
and lipids pinnatifida 37 °C, pH 6.2 (2013)
a-Amylase Polysaccharides Panax ginseng – Sun et al. (2015)
Pectinase and Carotenoids Tomato waste – Strati et al. (2014)
cellulase
Lipase and Proteins Olive pulp and Temperature Vergara-Barberán
phospholipase stone 30–40 °C, pH et al. (2014)
7.0, time
15 min
Papain, Fatty acids Strongylocentrotus Temperature Zhu et al. (2010)
protease, and nudus 40–55 °C, pH
trypsin 7.8–8.5, time
180 min
during the extraction process of oils from oil seeds. Carbohydrases hydrolyzes the
cell wall and enables higher release of oil in aqueous media, whereas proteolytic
enzymes improve the yield of oil by hydrolyzing the structural fibrous protein in
which fat globules are embedded (Yoon et al. 1991). Moreover, the proteolytic
enzyme modifies the emulsifying capacity of protein released in aqueous media.
The proteolytic enzymes used in the process of extraction have a huge impact on
the emulsifying capacity of the released proteins. The emulsifying capacity of the
protein increases during enzymatic proteolysis until certain degree of hydrolysis is
achieved, and thereafter, it starts decreasing. However, the reverse happens to the
stability of resulting emulsion (Puski 1975). The proteolytic enzymes used in
the extraction process can have positive or negative effect on process depending on
the degree of hydrolysis of the protein. The proteolytic enzyme releases the oil from
lipid bodies, but at the same time, the increased emulsifying capacity can lower the
extraction of free oil. This makes it necessary to control the process and extent of
proteolytic action to obtain a higher oil yield.
The cell walls and cuticles of marine algae are made up of chemically complex
and heterogeneous biomolecules. It needs carbohydrases and proteases to break
down the cell walls of natural matrices to release the cell content (Grosso et al.
2015). Some commonly used enzymes for these applications are xylanase, arabi-
nase, cellulase, amylase, protease, and glucanase (Kadam et al. 2013). Brown
seaweed Undaria pinnatifida contains alginate polysaccharide in abundance as a
part of cell wall and intracellular materials. In this case, alginate lyase was used to
increase the extraction of fucoxanthin from the seaweeds. Alginate lyase degrades
the alginate by b-elimination mechanism targeting glycosidic linkages between
monomers (Billakanti et al. 2013).
Presently, it appears that the aqueous extraction process using enzymes are
gaining attraction majorly for oils with high commercial value such as olive oil and
avocado oil. However, the cost of enzyme-assisted processes needs to be taken into
consideration which is mainly due to the separation steps, water and enzyme recycle
process, and reutilization.
3.2 Advantages of Enzyme-Assisted Extraction
1. This technology gives higher yields by breaking down the complex structure of
raw material.
2. It removes unwanted components of raw material selectively.
3. It shows high catalytic efficiency and preserves the original efficacy of natural
products.
4. It reduces the time of extraction and volume of solvent used.
4 Enzyme-Assisted Extraction of Bioactives

from Natural Sources
Bioactives such as oils, proteins, carbohydrates, and phenolics can be obtained from
variety of sources such as plants, bacteria, fungi, algae, and animals. The specific
details of enzyme-assisted extractions vary with the biomolecules of interest and its
source material.
4.1 Oils
Plant oils are commonly used in food, detergent, and paint industries. Plant oils
with higher content of polyunsaturated fatty acids (PUFAs) are important in food
industries. Conventionally, plant oils have been extracted using solvent extraction
(Bernardini 1973) where hexane is a commonly and commercially used solvent

(Rosenthal, Pyle and Niranjan 1996). However, hexane causes many environmental
concerns. Hence, aqueous extraction methods are better alternatives to organic
solvent extraction. Although aqueous extraction is an environmentally cleaner
technique, it is not successful due to the lower oil yields (Rosenthal et al. 1996).
This limitation can be overcome using enzymatic treatment during aqueous ex-
traction of oils (Badr and Sitohy 1992). Moreover, it is also beneficial to simul-
taneous extraction of oils and proteins (Jiang et al. 2010; Hanmoungjai et al. 2002).
4.1.1 Structure of Oleaginous Plant Seeds
The major oil and protein content of oilseeds is found in discrete cellular organelles
called lipid and protein bodies or oleosomes and aleurone grains, respectively.
Scanning electron microscope (SEM) analysis reveals the oleosomes from soybean
(Wolf and Baker 1975) and peanuts (Young and Schadel 1990) to be embedded in a
cytoplasmic network composed of proteins. The oleosomes and cytoplasmic net-
work fill up the spaces between protein bodies (Young and Schadel 1990). The
cytoplasmic network thus contains proteins and lipids. On the other hand, the walls
surrounding the cells are predominantly made up of cellulose, hemicellulose, lignin,
and pectin. It is significantly notable that the oleosomes contain high amount of
proteins called oleosins that form a membrane around the oleosomes, which play a
role in stability of these bodies.
4.1.2 Process of Enzymatic Extraction of Oils
Extraction of oils using enzymatic extraction process includes preliminary steps

similar to the conventional extraction method such as cleaning, cracking, flaking,
and pressing. The pressed oleaginous material can be treated with enzymes before
further step. Enzymes such as cellulases and pectinases are used either individually
or in combination. The use of combination of hydrolytic enzymes is however
preferred due to better yields so obtained. The enzymatic pretreatment is followed
by the conventional solvent extraction method to obtain higher yield of oils
(Domínguez et al. 1994).
Mechanical pretreatment processes such as flaking and extrusion impact on both
the aqueous oil process (AEP) and enzyme-assisted aqueous oil extraction process
(EAEP) (Lamsal et al. 2006). The oil extraction is reportedly increased from 46 to
71% in AEP and 56 to 88% in EAEP after including extrusion step in the extraction
process. However, extrusion denatures the protein and poses a challenge for the
simultaneous recovery of proteins (Caine et al. 1998). Enzyme-assisted extraction
processes for oils have been well reported in the literature (Moura et al. 2008;
Domínguez et al. 1994; Latif et al. 2008). However, there are some stringent
conditions that need to be maintained for enzyme-assisted aqueous extraction
process, which include the following:
1. Reduction in particle size of the source material to facilitate proper enzymatic

action and
2. The control over moisture content for effective enzyme action. Enzymes such as
cellulase need control over the moisture content as water activity plays a sig-
nificant role in the swelling and expansion of fiber and also in increasing the
surface area for activity of the enzyme (Domínguez et al. 1994).
Latif and Anwar (2011) used enzyme-assisted aqueous extraction to obtain oil
and protein from sesame seeds using a mixture of enzymes including protease and
carbohydrases (cellulase, hemicellulase, xylanase, b-glucanase and arabinase). This
extraction technique not only enhanced the yield of oil but also improved the
quality of oils extracted. Oxidative stability, antioxidant activity, and tocopherol
profile of sesame seed oil obtained after enzymatic extraction were better than that
obtained after hexane extraction. Though enzyme-assisted extraction is commonly
carried out using aqueous extraction, some attempts have been made using methods
other than aqueous extraction. The seeds of rose hip have been used to extract oil
using enzyme-assisted cold pressing (Concha et al. 2004). Enzymatic pretreatment
using pectinase, cellulase, and glucanase enhanced the oil extraction rate as well as
the yield of oil. In the aqueous extraction method, enzyme degrades the seed cell
wall and ruptures the polysaccharide–protein colloids. It forms an emulsion that
lowers the yield. However, in the absence of protein–polysaccharide colloids as is
the case in cold pressing, enzyme facilitates only hydrolysis of seed cell wall. The
enzyme-assisted cold pressing has also been reported to extract hemp seed oil (Latif
and Anwar 2009) with significant improvement in the oil yield. Apart from higher
oil yields, the method stabilizes the other seed constituents such as proteins and
fiber. In addition, the color intensity, tocopherol levels, rancimat profile, and sen-
sory score are better than conventionally extracted control samples.
Enzymatic pretreatment has also been used to assist the alkaline extraction
method to obtain oil from rapeseeds (Zhang et al. 2007). In this process, rapeseed
slurry is first treated with pectinase, cellulase, and b-glucanase and then subjected to
alkaline extraction and alkaline protease hydrolysis to produce protein hydrolyzate
and de-emulsify the oil. Many similar reports are available in the literature showing
extraction of oil from plant sources using enzyme-assisted extraction process.
Table 2 shows the oils extracted from various plant sources using enzyme-assisted
extraction.
4.1.3 Phenolics
Phenolics are secondary metabolites possessing one or more aromatic rings with
one or more hydroxyl groups. A vast variety of phenolics are known till date which
are structurally as simple as phenolic acids or as complex as highly polymerized
tannins (Dai and Mumper 2010). Researchers and food manufacturers have special
interest in this class of bioactives due to their potent antioxidant properties,
abundance in the diet, and ability to prevent various oxidative stress-associated
Table 2 Enzyme-assisted extraction of oil from different plant sources

Source (oilseed) Enzyme used Concentration of Yield Reference
enzyme/activity (%)
Soybean Protease 0.2% 86.0 Yoon et al.
(1991)
Soybean Protease 0.5% 97 Campbell
(2010)
Rapeseed Pectinase + cellulase 0.4:0.1% 80.2 Deng et al.
(1992)
Peanut Protease 3% 78.0 Lanzani et al.,
1975
Peanut Protease 1.5% 79.32 Jiang et al.
(2010)
Onion Amylase + cellulase 1:1% 18.38 Salina et al.
(2013)
Sesame Protease 2% 57.4 Latif and
Anwar (2011)
Canola (Brassica napus Carbohydrase – 26.0 Latif et al.
L.) (2008)
Goldenberry(Physalis Cellulase + pectinase 1:1 v/v 20.9 Ramadan
peruviana L.) pomace et al. (2008)
Rice bran Protease 1% 79 Hanmoungjai
et al. (2002)
diseases. It shows preventive action in case of many diseases such as cardiovas-

cular, neurodegenerative diseases, and cancer (Manach et al. 2004). In order to
utilize these bioactive for the preparation of dietary supplements or nutraceuticals,
food ingredients, pharmaceutical, and cosmetic products, they are required to be
efficiently extracted from raw materials.
Conventionally, solvent extraction method is common for the extraction of
phenolics from plants using solvents such as methanol, ethanol, acetone, and ethyl
acetate, either individually or in combination with different proportions of water.
Moreover, phenolics are generally attached to other plant components such as
carbohydrates and proteins which do not allow the development of a universal
protocol for the phenolic extraction. An additional step needs to be performed to
remove the unwanted components from the mixture after solvent extraction (Dai
and Mumper 2010). The bound phenolics can be extracted using pectinolytic
enzymes and cell wall-degrading enzyme preparation.
Continued research is being carried out using water as a sole extraction solvent.
However, the problem of lower yield persists in extraction of phenolics when
compared with that obtained by using solvents such as acetone (Meyer et al. 1998).
Solubility of phenolic compounds increases with an increase in the polarity of
solvent used (Naczk and Shahidi 2006). However, too high water content in
extraction solvent causes collateral extraction of other phytochemicals, thereby
lowering the concentration of phenolics (Spigno et al. 2007).
The mode of action of hydrolytic enzymes on extraction of phenolics is by

cleaving the cell wall components, thus favoring the exposure of phenolics to the
extraction. Li et al. (2006) observed a 28% increase in concentration of phenolic
compounds extracted using commercial enzyme Cellzyme MX, as compared to the
control (without enzyme). Also, Hansen and Laroze (2009) observed a 35%
increase in the yield of phenolics, while evaluating the effect of enzymatic treatment
during hydroethanolic extraction from raspberry residue.
As explained in earlier sections, the extraction efficiency may vary with several
processing variables such as type of enzymes used, substrates, solvents, and
operating temperature (Meyer et al. 1998). The extraction yield depends signifi-
cantly on particle size of mashed samples. The yield of phenolic compounds can be
increased by lowering the particle size (Pinelo et al. 2005). Salina et al. (2013)
studied enzyme-assisted extraction of phenolics from onion using different enzymes
(cellulase and amylase individually and in combination). The maximum phenolic
content of 5.9 mg/ml was obtained using cellulase individually for 16 h at 35 °C.
Cellulase has also been used for the extraction of total phenolics from citrus peels
(Li et al. 2006). A 65.5% recovery of phenolics with potential antioxidant activity
was observed. Commercial enzymes, Viscozyme L (carbohydrases including ara-
binase, cellulase, b-glucanase, hemicellulase, and xylanase) and Rapidase, had been
used to extract the phenolic compounds from cauliflower harvest and showed the
combination of both the enzymes to result in better efficiency in recovery of phe-
nolics (Huynh et al. 2014). Furthermore, pectinase was more useful to extract
phenolic antioxidants from raspberry wastes (Laroze et al. 2010). Experimental
results showed that the enzyme-assisted extraction using hydroethanolic mixture for
18 h at 50 °C could efficiently increase the phenolic content to 35%.
One of the most crucial properties of plant phenolics is to retain the antioxidant
activity. In vitro assays have shown plant phenolics to be a more potent antioxidant
than vitamin C, vitamin E, and carotenoids (Rice-Evans et al. 1995, 1996). This
makes the extraction of phenolics from plant material important. Several reports are
available in the literatures (Table 3) on extraction of phenolics from a variety of
sources using enzyme-assisted extraction.
4.1.4 Flavorings and Colorants
The use of colorants and flavorings in food industry is increasing significantly since
the last few decades. The quality of food is majorly affected by color and flavor
which decides the appearance and acceptance of the product. The growing demand
for colors is met by synthetic colorants. However, the synthetic colorants are prone
to have adverse health effect including carcinogenicity. Hence, these are being
phased out by regulatory bodies in many countries. This in turn has led to a growing
interest to find natural food colorants and flavorings (Chandrasekaran 2012).
Methods such as solvent extraction, hydrodistillation, steam distillation, and
supercritical carbon dioxide extraction are common for the extraction of flavorings
and colorants. Generally, the extraction of flavorings and colorants is incomplete
Table 3 Enzyme-assisted extraction of plant phenolics from various plant sources
Source Phenolic Enzyme Enzyme Yield of Reference
extracted concentration phenolics
Blackberry Anthocyanins Pectinase 0.2% 639 g/l Hankun et al. (2014)
juice
8 Enzyme-Assisted Extraction of Bioactives
Saffron tepals Anthocyanin Cellulase + hemicellulase + pectinase 5% 6.7 mg/g Lotfi et al. (2015)
Grape pomace Phenolic acids Pectinase + cellulase 2:1 91.9% Maier et al. (2007)
Grape pomace Total phenolics Pectinase 10% 3072 mg/l Meyer et al. (1998)
Apple skin Total phenolics Cellulase + pectinase + protease 1:1:1 104.94 mg/l Pinelo et al. (2008)
Grapes Total phenolics Pectinase – 90 ± 0.37% Gómez-García et al.
(2012)
Unripe apples Total phenolics Cellulase – 7.08% Zheng et al. (2009)
181
because cellulose is responsible for its sequestration in the cell. Enzymes cause
partial destruction of plant cell wall and help in separation of intracellular com-
pounds (Waliszewski et al. 2007).
Cellulolytic enzyme has been used to extract vanillin from vanilla beans by
Waliszewski et al. (2007). They found the hydration process in 5% ethanol for 48 h
and enzymatic pretreatment with cellulase for 12 h to double the vanillin content in
the extract with excellent sensory quality as compared to control treatment without
enzymes. Zhang et al. (2014) also attempted to extract vanillin from green vanilla
pods using enzyme-assisted extraction combined with prefreezing and thawing.
Cellular compartmentalization of vanilla green pods was destroyed by prefreezing
and thawing. It was followed by treatment of pectinase to hydrolyze the pectin
between glucovanillin substrate and b-glucosidase. This method could successfully
transform the glucovanillin to vanillin and produce natural vanillin from green
vanilla.
The extracted anthocyanins find wide range of applications in various industries.
They have been used as natural food colorants and antioxidants in pharmaceutical
products. Extraction of anthocyanins using different enzymatic and non-enzymatic
extraction methods has been studied by various researchers (Chandrasekhar et al.
2012; Vanini et al. 2009; Hankun et al. 2014). Lotfi et al. (2015) used pectinex
(containing cellulase, hemicellulase, and pectinase) at varying concentrations to
extract anthocyanins from saffron tepals and also compared the yield with con-
ventional ethanol extraction method. They observed a 40% increase in the yield of
total anthocyanins.
Barzana et al. (2002) extracted carotenoids from marigold flowers using enzy-
matic extraction with hexane as a solvent. Under optimal conditions, they obtained
a recovery yield of 97%. Lenucci et al. (2015) performed studies on enzymatic of
lycopene, a carotenoid red pigment, synthesized and stored in tomato berry chro-
moplasts using glycodiase. The results showed that the enzymatic extraction could
increase the yield of lycopene by 153% as compared to solvent extraction.
4.1.5 Carbohydrates
Carbohydrates play an important role in cell signaling, cell adhesion, and molecular
recognition in the immune system (Dwek 1996). Researchers have shown various
biological activities of carbohydrates such as wound healing, stimulation of
immune system, and treatment of tumor (Schmidgall et al. 2000). Hence, studies are
being performed to isolate and identify carbohydrates from different plant sources
and also to test their pharmacological activities. Carbohydrates are ubiquitously
present in plants and can be isolated from different parts of plant such as leaf, seed,
and root, each of which may give carbohydrates with differential bioactivity.
The conventional method for extraction of carbohydrates from plants involves
steps such as size reduction, extraction, and filtration. However, aqueous extraction
is a safe and economical method which gives better yield of carbohydrates than
conventional solvent extraction methods (Hu et al. 2013). Apart from various
advantages of aqueous extraction method listed in earlier sections, there are other
benefits of using water such as easy penetration in the plant tissue resulting in high
yield and stability of carbohydrates extracted (Hu et al. 2013). Hence, recent studies
have focused on enzyme-assisted extraction of carbohydrates from plants. The
general method for enzyme-assisted extraction of carbohydrates is similar to the
conventional extraction method. The dried plant material is powdered and extracted
using water in the presence of enzyme. Proteins can be removed with lead acetate,
and excess lead can be removed with potassium oxalate. Filtration can then be
carried out to eliminate residual material (Weinmann 1947).
Smith, Paulsen and Raguse (1964) studied the method of enzyme-assisted
extraction to obtain carbohydrates from grass and legume tissues using takadiastase
and obtained yields better than the aqueous extraction. Takadiastase hydrolyzes
starch (insoluble in water) into maltose residues (soluble in water), which increases
the total carbohydrate yield. Bahramian et al. (2011) showed the significance of
enzyme dosage during the study of optimization of enzyme-assisted extraction of
sugars from kabkab date fruit using pectinase and cellulase. Furthermore, Patindol
et al. (2007) used cellulase to extract oligosaccharides by enzyme-assisted extrac-
tion method. They could improve the total carbohydrate yield from 69.2 to 87.2%.
Ng et al. (2014) used enzyme-assisted extraction method to obtain cellobiose,
glucose, and fructose by hydrolysis of agricultural waste grapefruit peel and orange
peel using cellulase. Besides cellulase, pectinase is also common for
enzyme-assisted extraction method to obtain carbohydrate of interest. This enzyme
has been explored by Dzogbefia et al. (2008) to extract starch from cassava with
high yield. Pectinolytic enzymes are used in the treatment of plant materials for cell
wall disintegration, de-pectinization, reducing viscosity to increase the flow rate,
and release cell components, thus increasing the ultimate yield (Demir et al. 2001;
Rai et al. 2004).
The method of extraction can be decided based on intention of application of
polysaccharides. This has been explained well from the experiment done by Pan
et al. (2015). They found that the polysaccharide obtained from Dendrobium
chrysotoxum using cellulase-assisted extraction had higher immunomodulatory
activity compared to that of hot water extraction method which showed better foam
stabilization activity. The improvement in the yield of polysaccharide extraction by
enzyme-assisted extraction method compared to hot water extraction method has
also been shown by Zhu et al. (2014) during the extraction of Hericium erinaceus
polysaccharides.
Although cellulase, pectinase, hemicellulase, and glucanase are commonly used
enzymes in enzymatic extraction of carbohydrates, other uncommon enzymes have
also been explored by some researchers based on the nature of raw materials used.
Chen et al. (2014) have used glucose oxidase for the extraction of polysaccharides
from Astragalus membranaceus. They have optimized the process by response
surface methodology and obtained a 250% increase in the yield and better
antioxidant activity as compared to control process. The same enzyme was also
used for the extraction of Fructus mori polysaccharides (Deng et al. 2014) and
found to increase in the yield of the polysaccharide by 160% using enzyme-assisted

extraction method.
4.1.6 Proteins
Among all the bioactives, proteins are most important as a nutritional and dietary
supplement. Proteins and peptides together contribute major constituents of regular
food and can be obtained from plant as well as animal sources. Various methods of
extraction and fractionation of protein and peptides are available, but the choice of
method depends on several factors such as solubility, hydrophobicity, molecular
weight, and isoelectric point (pI). Efficient and optimized techniques must be used
to remove interfering compounds such as lipids, phenolics, carbohydrates, oxidative
enzymes, and pigments without protein degradation or modification.
The presence of indigenous proteases in plant tissue makes the extraction of
proteins complicated (Wang et al. 2008). Proteins are usually found in protein
bodies (also called as aleurone grains) inside the cells. Hence, the complete solu-
bilization and extraction of proteins depends on cell disruption. The method used
for cell disruption depends on the types of plant material used (leaf, fruit, root, seed,
etc.) or even on the stage of development of plant. A number of chemical methods
(using solvents such as ether, acidified alcohol, and chloroform) and physical
methods (such as bead-beating, sonication, and mortar–pestle) are known to be used
for the disruption of cells. However, due to the differences in the nature and
proportion of components, the choice of method may vary.
Commercially produced protein concentrates usually consist of aqueous solu-
bilization of protein, thus making water as a solvent of choice for extraction. The
extraction yield of protein can further be increased by using enzyme-assisted
aqueous extraction of proteins. Different carbohydrases can be used to release
proteins from raw materials. Guan and Yao (2008) used viscozyme L to hydrolyze
cell wall by cleaving the linkages within polysaccharides that effectively release
intracellular protein from oat bran. Jung et al. (2006) showed successful use of
pectinase to improve extractability of soy protein without protein degradation. The
protein recovery was increased by 50% as compared to control with improved foam
stability. Further, Vergara-Barberán et al. (2015) explored the use of cellulase to
improve the protein extraction from olive leaves. They optimized the process of
enzyme-assisted extraction of proteins from olive leaves and found that the method
was faster with higher recovery and reduced solvent usage. Recently, the focus has
been shifted to use proteases to hydrolyze the proteins partially and convert them to
peptides. This increases the solubility of peptides making their extraction effective.
Oil seed meal that is obtained as by-product after meal production is a potential
source of protein. Proteases have been used to extract proteins from oilseed meals
such as rapeseed, soybeans, and microalgae meals (Sari et al. 2013). The addition of
proteases enhances the yield of protein extraction to 90% from soybean meals and
50–80% from rapeseeds and microalgae meals. The use of proteases has also been
Table 4 Enzyme-assisted extraction of proteins from different plant sources

Source Enzyme used Concentration of Yield Reference
enzyme/activity (%) (%)
Peanut Protease 1.5 71.38% Jiang et al. (2010)
Sesame Protease 2 87.1% Latif and Anwar
(2011)
Soybean Protease 1 87% Campbell (2010)
Rice Protease 2 68% Hanmoungjai et al.
bran (2002)
Soybean Protease 0.5 85% Moura et al. (2008)
Rapeseed Protease 1.5 83% Zhang et al. (2007)
Rice Phytase + xylanase – 74.6% Wang et al. (1999)
bran
explored for the extraction of peptides of interest for various pharmacological

actions. Furthermore, antioxidant and a-amylase inhibitory peptides have been
extracted from pinto beans by enzyme-assisted extraction using enzyme protamex
(Ngoh and Gan 2016). Similarly, antioxidative and antihypertensive bioactive
peptides have been successfully extracted from Parkia speciosa seed using alcalase
(Siow and Gan 2013).
Simultaneous recovery of protein and lipids using enzyme-assisted extraction
method is gaining attraction due to dual benefits. Protease has been used for
simultaneous recovery of protein and oil from extruded soybean flakes using
enzyme-assisted aqueous extraction method (Moura et al. 2008). The yield of oil
was 96%, whereas that of protein was 85%. Niu et al. (2012) used the same
technique for extraction of rapeseed oil and protein from dehulled cold-pressed
double-low rapeseed cake. They obtained 82.10% yield of protein and 71.89%
yield of oil using enzymes Viscozyme L (carbohydrases) and Alcalase (proteases).
The reports available on enzyme-assisted extraction of protein from various
plant sources are compiled in Table 4.
5 Bioactives from Non-plant Sources
Although plants are the most important sources of bioactives, other sources such as
bacteria, algae, fungi, and animals have also been explored to obtain bioactive of
interest. This reduces the dependency on plant sources. The bioactives obtained
from different sources using enzyme-assisted extraction method are detailed in the
subsequent sections.
5.1 Oils
Since centuries, the major sources of oils are from plants and used mainly for
human consumption, which then extended in recent times for biodiesel production.
Currently, oils derived from microbial sources (single cell oils) are gaining
importance as an alternative to vegetable oil for biodiesel production mainly due to
their comparable chemical properties (Christophe et al. 2012). All the oils that are
presently being produced as single cell oils are high in PUFA content and are
intended to be used mainly for human consumption as nutraceuticals. Some other
oils are also being used as feed for animals and farmed fish (Ratledge 2013).
The growing demands for energy resources have caused an adversity, and hence,
the current research on biodiesel production focuses mainly on microbial oils (Xia
et al. 2011). Microalgae are also gaining attention due to their ability to produce
high amounts of oil and fast growth, by fixing large amounts of CO2 (Demirbas and
Demirbas 2010; Huo et al. 2011). Conventional methods used for the extraction of
oils require the algal or fungal biomass to be in dry form. This needs an additional
step of dehydration during extraction which further increases the process cost.
Moreover, conventional extraction methods also use chemical solvents which are
hazardous to the environment and also give inadequate recovery, thus making their
use undesirable. As detailed in earlier sections, the use of green extraction methods
such as enzyme-assisted extraction overcomes these drawbacks for better end
application. Enzyme-assisted extraction is not only environmentally friendly but
also economically efficient method, as it avoids the additional drying process and
also reduces the use of hazardous solvents during extraction process (Liu et al.
2013). Moreover, it does not affect the quality of value-added biomass
(Gómez-García et al. 2012) making it desirable.
Algal and fungal cell walls resemble plant cell wall and also act as a barrier to
the extraction of bioactives using enzyme-assisted extraction. For efficient extrac-
tion, a combination of enzymes can be used which effectively breaks the microbial
cell walls. Several researchers have studied the effect of combination of enzymes on
extraction of oil from algae (Huo et al. 2015; Liang et al. 2012). Algal cell walls are
made up of either polysaccharides such as cellulose or a variety of glycoproteins, or
both. This makes the cellulases and proteases good candidates for lysis of algal cell
walls. Huo et al. (2015) studied the effect of different quantities of enzymes (cel-
lulase, pectinase, and hemicellulase) taken in combination with each other, along
with the process parameters (temperature, pH, algal biomass concentration) on the
extraction of oil from wet microalga Scenedesmus sp. G4. They observed the
extraction yield reaches 86.1% under optimal conditions. Liang et al. (2012) also
carried out enzyme-assisted extraction of lipid from microalgae using enzymes
cellulase, snailase, protease (neutral and alkaline), and trypsin. They observed a
highest lipid recovery of 49.82% by using enzyme treatment along with sonication
at pH 4 with enzymes, snailase and trypsin being better than cellulase and proteases.
Zuorro et al. (2015) evaluated enzyme-assisted extraction of lipids from microalgal
cells using 1,4-ß cellobiosidase, galactomannanase, and ß-glucosidase and recov-

ered 70% of the lipids.
The concentration of enzyme(s) and their types may vary depending upon the
microbial species. After the cell lysis using hydrolytic enzymes, hexane is added to
the enzymatically hydrolyzed algal biomass as an extraction solvent, and the
mixture is then centrifuged. This gives two phases, viz. a light phase, which pre-
dominantly contains the extracted oil in solvent, and a heavy phase that contains
water and algal residue (along with oil and solvent carryover). The two phases can
be separated, and oil can be isolated from light phase, thus leaving solvent (hexane)
behind. The solvent can further be reused for next extraction cycle. Subsequently,
the residual material can be sent for anaerobic digestion. Water can also be used as
an alternative to hexane, thus making the process cheaper and environmentally
friendly. It forms two phases comprising of extracted oil in one phase and the water
with the residual biomass in another (Davis et al. 2013).
Several fungal species have the capacity to produce and accumulate high
amounts of lipid (up to 70%). These oleaginous fungi produce oils that contain
triacylglycerol (TAG) and PUFA and hence are desirable for applications in the fuel
and food industries. In fungal biomass, typically, the extraction of oil is carried out
using Soxhlet extraction and pressurized liquid extraction (Cescut et al. 2011).
Methods for extraction of lipids using wet cell mass by organic solvents (Bligh and
Dyer 1959) and supercritical fluids (Halim et al. 2011) have also been established.
Fungal cell walls are made up of components such as mannoproteins, ß-glucan, and
chitin. Fungal chitin is difficult to degrade as compared to plant cellulose. Thus, the
choice of enzyme here is different as compared to enzymes used during the
extraction of bioactives from plant/algal cells. Hydrolytic enzymes such as chitinase
and ß-1,3-glucomannanase can be used for breaking fungal cell walls. Breaking the
cell wall makes lipids accessible to the extraction solvent that improves the
extraction efficiency.
Jin et al. (2012) extracted lipids from the culture of yeast Rhodosporidium
toruloides. They used the recombinant ß-1,3-glucomannanase for this purpose; then
carried out the extraction in ethyl acetate; and found 96.6% lipid to extracted
directly from culture without dewatering. Thus, the process was deemed as
cost-effective.
5.2 Polysaccharides
Several algal, fungal, and bacterial species have also been proven to be a potential
source of polysaccharides. Extensive research has been done to study the extraction
methods and biotechnological applications of these polysaccharides (Costa et al.
2010; Rioux et al. 2007; Wijesinghe et al. 2011). Fu et al. (2010) studied the
enzymatic hydrolysis of microalgae cell walls using immobilized cellulase under
the optimized conditions to isolate reducing sugars. Wijesinghe et al. (2011) also
carried out enzyme-assisted extraction of sulfated polysaccharides from the brown

seaweed Ecklonia cava.
5.3 Phenolics
Few attempts have been made to extract phenolics from other than plant source.
Machu et al. (2015) performed experimental studies on the extraction of phenolics
such as gallic acid, 4-hydroxybenzoic acid, catechin hydrate, epicatechin, catechin
gallate, epicatechin gallate, and pyrocatechol from commercial algal foods such as
brown and red algae, green freshwater algae, and blue green algae using different
extraction solvents. The lysis of microbial cells can be carried out using hydrolytic
enzymes to improve the extraction of phenolics. The effect of hydrolytic enzymes
such as proteases and carbohydrases on the lysis of algal cells and improvement in
the extraction yield of polyphenols and other antioxidant ingredients from red algae
Palmaria palmate were studied by Wang et al. (2010). The protease could enhance
the extraction of polyphenols and other active components as compared to carbo-
hydrases and water extraction. Heo et al. (2005) also showed the antioxidant
activities of brown seaweed extracts obtained after enzymatic process to be higher
than the commercial antioxidants.
6 Combination of Enzyme-Assisted and Other

Techniques to Extract Bioactives
With the rising importance of enzyme-assisted extraction of bioactives 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 bioactives. Non-conventional techniques such as
three-phase partitioning, microwave-assisted extraction, ultrasound-assisted
extraction, and supercritical fluid extraction have already been studied (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 bioactives.
6.1 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 several 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 used directly on crude plant materials, thus reducing
the process cost. The use of inexpensive chemicals such as t-butanol and ammo-
nium sulfate also makes the process economical (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 h. 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 en-
zymes 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.
6.2 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 extrac-
tion (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.
6.3 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.
6.4 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. Table 5 shows the extraction of bioactives
using combination of enzyme-assisted and other techniques.
7 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 syner-
gistically 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 temper-
atures, 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
192
Table 5 Combination of enzyme-assisted extraction and other techniques for the extraction of different bioactives from various sources
Technique Enzyme(s) used Bioactive(s) of Source Yield Reference
interest
Enzyme-assisted three-phase ProtizymeTM Oil Soybean 98% Gaur et al.
partitioning (EATPP) (2007)
Enzyme-assisted three-phase a-Amylase Oleoresin Turmeric 8.96% Kurmudle
partitioning (EATPP) et al.
(2011)
Enzyme-assisted supercritical Enzyme cocktail (acid cellulase, Phenolics Pomegranate 32.19 ± 1.26% Mushtaq
fluid extraction pectinase, viscozyme, kemzyme, et al.
alcalase) (2015)
Microwave-assisted enzymatic Cellulase Phenolics Geranium 6.79 mg/g (corilagin) Yang et al.
extraction (MAEE) (corilagin and sibiricum and 19.82 mg/g (2010)
geraniin) (geraniin)
Microwave-assisted enzymatic Cellulase Polyphenols Waste 1.75 ± 0.06% Zhang
extraction (MAEE) peanut shells et al.
(2013)
Microwave-assisted aqueous Enzyme cocktail (cellulase, pectinase, Oil Pumpkin 64.17% Jiao et al.
enzymatic extraction and proteinase) seeds (2014)
(MAAEE)
Ultrasonic-assisted enzymatic Pectinase Flavonoids– Celery 42.5 mg/g (Luteolin) Zhang
extraction (UAEE) luteolin and and 25.3 mg/g et al.
apigenin (apigenin) (2011)
Ultrasonic-assisted enzymatic Papain Polysaccharide Zizyphus 21.95% Sun et al.
extraction (UAEE) jujuba (2011)
Ultrasonic-assisted enzymatic Enzyme cocktail (papain, pectase, Crude Epimedium 5.98% Chen et al.
extraction (UAEE) cellulase, and a-amylase) polysaccharides leaves (2012)
S.J. Marathe et al.
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;
(v) 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).
8 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 asso-
ciated with cost-effective applications in current commercial processes. The pos-
sible solutions for further commercialization of enzymes in extraction industry
include the following: (a) reduction in the cost of enzyme production, (b) im-
provement 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 engi-
neering approaches. However, a technoeconomic 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, stor-
age, and shipping and could reduce the production costs by using pretreated sub-
strates already available at the biorefinery (Culbertson et al. 2013).
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Chapter 8

Enzyme-Assisted Extraction of Bioactives

Sandesh J. Marathe, Swati B. Jadhav, Sandip B. Bankar

S.J. Marathe S.B. Jadhav R.S. Singhal (&)

© Springer International Publishing AG 2017 171

depends on the type of raw material, environmental concerns, process conditions,

2 Conventional Extraction Techniques

2.1 Solvent Extraction Methods

higher molecular weight polyphenols have shown preference toward aqueous

2.2 Physical Extraction Methods

Enzyme-assisted extraction can be considered as a boon to overcome the limitations

3.1 Mechanism of Action of Enzyme

The mechanism of enzymatic action is the heart of enzyme-assisted extraction

3.2 Advantages of Enzyme-Assisted Extraction

4 Enzyme-Assisted Extraction of Bioactives

(Bernardini 1973) where hexane is a commonly and commercially used solvent

4.1.1 Structure of Oleaginous Plant Seeds

4.1.2 Process of Enzymatic Extraction of Oils

Extraction of oils using enzymatic extraction process includes preliminary steps

1. Reduction in particle size of the source material to facilitate proper enzymatic

Table 2 Enzyme-assisted extraction of oil from different plant sources

diseases. It shows preventive action in case of many diseases such as cardiovas-

The mode of action of hydrolytic enzymes on extraction of phenolics is by

4.1.4 Flavorings and Colorants

found to increase in the yield of the polysaccharide by 160% using enzyme-assisted

Table 4 Enzyme-assisted extraction of proteins from different plant sources

explored for the extraction of peptides of interest for various pharmacological

5 Bioactives from Non-plant Sources

cells using 1,4-ß cellobiosidase, galactomannanase, and ß-glucosidase and recov-

carried out enzyme-assisted extraction of sulfated polysaccharides from the brown

6 Combination of Enzyme-Assisted and Other

With the rising importance of enzyme-assisted extraction of bioactives from plant

6.1 Enzyme-Assisted Three-Phase Partitioning (EATPP)

Three-phase partitioning (TPP) is a new technique used to separate proteins by

6.2 Microwave-Assisted Enzymatic Extraction

Microwaves are electromagnetic radiations with their frequencies ranging from

6.3 Ultrasound-Assisted Enzymatic Extraction

6.4 Enzyme-Assisted Supercritical Fluid Extraction

7 Large-Scale Enzymatic Processes

8 Challenges and Future Perspectives

Extensive research has been carried out in large-scale application of enzyme-

be made for better commercial applications. Another novel approach is expressing

Chandrasekhar J, Madhusudhan MC, Raghavarao KSMS (2012) Extraction of anthocyanins from

Dzogbeﬁa V, Ofosu G, Oldham J (2008) Evaluation of locally produced Saccharomyces cerevisiae

Vergara-Barberán M, Lerma-García MJ, Herrero-Martínez JM, Simó-Alfonso EF (2014) Efﬁcient

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