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Enzyme	-Assisted	extraction	of
Bioingredients
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Synthite	Industries
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Karunya	University
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Special Report
213Chemical Weekly October 1, 2013
Enzyme-assisted extraction of bioingredients
I
n the modern world, bioingredi-
ents have an important role to play
in different spheres of human life.
They can be a major component of
food we eat; beverages that quench our
thirst; medicines that cure our illness;
cosmetics that make us beautiful and
clothes that make us fashionable. So the
demand for bioingredients is increasing
day by day. Modern science and tech-
nology is being challenged by the rising
demand from society for development
of new bioingredients, sources and
method of isolation.
Bioingredients are the active prin-
ciples of plants and are generated in the
plants either by the primary or second-
ary metabolic reactions taking place
during the course of plant growth. The
primarymetabolitesofplantsaremainly
lipids, protein and carbohydrates. The
secondary metabolites are classified
into three main groups: phenolics, ter-
penoids and alkaloids.
As a consequence of the increased
demand for bioingredients like essential
oil, natural colours, pure components
from the industries, there is a need for
extensive collection of information on
the composition of bioingredients and
their recovery methods.
The extraction of bioingredients
from plant material can be achieved by
a number of different methods, like:


 	Expression;


 	Distillation (hydro- or water-distil-
lation, water and steam distillation,
steam distillation);


 	Solvent extraction;


 	Super Critical Fluid Extraction
(SCFE); and


 	Enzyme assisted extraction.
For each method there may be
many variations and refinements and
the extraction may be conducted under
reduced pressure (vacuum), ambient
pressure or excess pressure. The choice
of extraction method will depend on the
nature of the material, the stability of
the chemical components and the speci-
fication of the targeted product.
Expression
Expression is used exclusively for
the extraction of citrus oil from the fruit
peel, because the chemical components
of the oil are easily damaged by heat.
Citrus oil production is now a major by-
product process of the juice industry.
Distillation
Distillation is a physical process
used in most of the industries to iso-
late the volatile portions of plant ma-
terials. The main advantage of distilla-
tion is that it can generally be carried
out with some very simple equipment,
close to the location of plant produc-
tion. Even in relatively remote loca-
tions large quantities of material can
be processed in a relatively short time.
There are various types of distillation
methods like hydro distillation, water
and steam distillation and steam distil-
lation. The selection of the distillation
method depends on the nature of the
plants and their parts.
Water distillation is the simplest
of the three distillation methods. It is
one of the oldest methods of distilla-
tion used. The plant material is mixed
directly with water in a still pot. A
perforated grid may be inserted above
the base of the still pot to prevent the
plant material settling on the bottom
and coming in direct contact with the
heated base of the still and resulting in
charring. The spice is fully immersed in
hot water. The result is a soup, which
carries aromatic molecules of the plant.
The method is not much in use these
days, because of the risk of overheating
K.C. BABY
Lead-Sales Application
Bio-Ingredients Division
Synthite Industries Ltd.
E-mail: babykc@synthite.com
DR. T.V. RANGANATHAN
Professor
Food Processing and Engineering
Karunya University, Coimbatore
E-mail: srivarahe@gmail.com
Special Report
Chemical Weekly October 1, 2013214
the plant and subsequent loss of the oil.
The water-distilled oils are commonly
darker in colour and have stronger still
‘off-note’ odours than oils produced by
other methods, and therefore tend to
be of the lowest value. Generally, this
method is followed at the laboratory
scale to screen the quality of the raw
material.
Hydro distillation is not commonly
practiced in industries because of the
long distillation time and the resulting
mass obtained after hydro distillation
is not easily amenable for oleoresin
extraction with solvents.
In steam-and-water distillation, the
basic still design is very similar to that
of water distillation. The plant material
is packed into the still pot sitting on a
grill or perforated plate above the boil-
ing water. The capacity of the still pot
volume is reduced, but it may be pos-
sible to achieve a high packing density
because the plant material is not sus-
pended in the water. The advantages of
steam and water distillation over water
distillation include:


 	Higher oil yield;


 	Oil component less susceptible to
change due to wetness and thermal
conductivity of the still from the
heat source;


 	The effect of refluxing is mini-
mised;


 	Oil quality is more reproducible;
and


 	Faster process, so more energy
efficient.
Steam distillation is the process of
distilling plant material with the steam
generated outside the still in a stand-
alone boiler. As in the steam-and-water
distillation system the plant material is
supported on a perforated grid above
the steam inlet. The powdered raw
material is charged into the still and
steam is introduced from the bottom of
the still. The steam carries off the vola-
tile components and passes through a
condenser where the volatiles get con-
densed and separated from water. Most
of the spices such as pepper, ginger,
cardamom, etc. are subjected to steam
distillation to obtain essential oils from
the spice.
Solvent extraction
Many herbs and spices cannot be
extracted by distillation method. In
such cases, solvent extraction is the
safest method for extracting high qua-
lity oil. In this process, the spices or
herbs are immersed in the solvent and
the ‘separation’ is performed chemi-
cally. These include pigments, volatile
molecules and non-aromatic waxes. A
solvent is a liquid or gas that dissolves
a solid, liquid or gaseous solute, resul-
ting in a solution. Solvents usually
have a low boiling point and evaporate
easily, or can be removed by distilla-
tion, leaving the dissolved substance
behind. Basically there are two cate-
gories of solvent, i.e., organic and in-
organic solvent. The selection of an
appropriate solvent is guided by theory
and experience. Generally, a good sol-
vent should meet the following criteria:


 	It should be inert to the reaction
conditions;


 	It should dissolve the reactants,
reagents and bio actives;


 	It should have an appropriate boil-
ing point; and


 	It should be easily removed at the
end of the reaction.
Spice oleoresins are prepared by
this method.
Super critical fluid extraction
(SCFE)
Solvent extraction is not considered
the best method for extraction as the sol-
vents can leave a small amount of resi-
due behind which could cause allergies
and affect the immune system. The most
recent method is that of the distinguished
carbon dioxide extraction. This method
uses carbon dioxide to extract the
essentialoilandoleoresinsfromtheplant
when liquefied under pressure. Once the
liquid depressurises, the carbon dioxide
returns to a gaseous state, and only pure
essential oil and resin remain. It employs
a much lower temperature than that of
steam distillation. This process is intend-
ed to introduce oils that remain close to
the way they reside in nature. This is pri-
marily due to the inert nature of the sol-
vent and lower pressures. There is some
disagreement to this theory in that due to
the acidic nature of CO2
one could argue
it is disruptive to the chemistry of the
resultant oil. Whereas terpenes are often
manufactured through the methodology
of distillation, lower terpene content is
produced through a CO2
extraction, a
higher ester range is manufactured, and
the addition of molecules far too large to
pass their way over through distillation
can be found. These findings confirm
that the oil does have more of the botan-
ics inherent personality.
SCFE has many advantages over
other methods of extraction like the
absence of solvent residues, no off-
odours, low mono-terpenes hydrocar-
bon levels, more top notes and more
back notes giving a full but different
profile than with traditional extraction
methods.
Special Report
215Chemical Weekly October 1, 2013
Enzyme assisted extraction
Modern science and technology
have helped flavour industries, food
industries, cosmetics and other non-food
industries to improve product qualities
by providing advanced equipments and
ingredients. To cop up with the emerg-
ing requirements, advanced extraction
technologies are required. Enzyme
assisted extraction is the recent approach
for extracting bioingredients from plant
materials. The applications of enzymes
in the extraction of essential oils from
oilseeds like sunflower, soybean, rape-
seed, corn, coconut, olives, avocado,
and also for the extraction of rice bran
oil etc. are well documented.
Enzymes are proteins with highly
specialised catalytic functions and pro-
duced by all living organisms. They
are responsible for many essential bio-
chemical reactions in microorganisms,
plants, animals and human beings.
Enzymes are essential for all metabolic
processes. Although like all other pro-
teins, enzymes are composed of amino
acids, they differ in function in that
they have the unique ability to facilitate
biochemical reactions without under-
going change themselves. This cata-
lytic capability is what makes enzymes
unique. In other words, they are highly
specific biological catalysts. Enzymes
not only work efficiently and rapidly,
they are also biodegradable. Enzymes
are highly efficient in increasing the
reaction rate of biochemical processes
that otherwise proceed very slowly, or
in some cases, not at all.
Enzymes are categorised according
to the compounds they act upon. Some
of the most common types include pro-
teases (which break down proteins), cel-
lulases (which break down cellulose),
lipases (which split fats into glycerol
and fatty acids), and amylases (which
break down starch into simple sugars).
For example, the enzyme lipase exerts
no action on starch substrate and amy-
lases do not act on the proteinaceous
substrate. This defines its ‘specificity’
and provides the basis of its classifica-
tion and name. Often, the trivial name
of the enzyme, derived from the trun-
cated substrate name with ‘ase’ added,
identifies the substrate or substrate
range better for food technologists than
does its systematic name or its Interna-
tional Union of Biochemistry Enzyme
Commission (IUB or EC) number.
Enzymes are classified into the fol-
lowing types: hydrolyzing enzymes,
oxidation-reduction enzymes, ligases,
group transfer enzymes, desmolases,
isomerizing enzymes, and carboxy-
lation enzymes. Based on their pro-
perty of catalysing definite reactions,
a particular enzyme acts on a specific
substrate.
The enzymes also do not become
part of the final product of the bio-
chemical reaction that they are catalys-
ing. When the biochemical reaction is
over, the product of the reaction leaves
the enzyme. The enzyme is then ready
to effect the same reaction on another
molecule again and again. Given the
right conditions to function, the enzyme
can go on and on for as long as needed.
Enzymes are large molecules with
hundreds of amino acids. Only a small
part of the enzyme participates in the
catalysis of biochemical reactions;
this is called the active site. The three-
dimensional structure of the enzyme
determines the appearance of the active
site. The active site accommodates the
shape of the biological substrate that
requires transformation. They fit like
a key in a lock. This is what makes
enzymes specific in their actions. Only
substances of the right shape will be
transformed. The specificity of action
of an enzyme on a specific substrate is
determined by the structure and confor-
mation of active site.
Enzymes have been used for hun-
dreds of years, and today the use of
them is almost without limits. The his-
torical uses of enzymes to make beer,
wine, cheese and bread are elegant
examples of the industrial exploitation
of the power and selectivity of enzymes.
In each case of enzyme application,
one has to standardise the operational
conditions viz., enzyme concentration
and time of incubation, temperature
of incubation, and optimum pH for the
maximum activity of the enzyme. To
use enzymes effectively in food appli-
cations, proper knowledge about the
nature of the enzyme, the source of the
enzyme, active site, mode of action,
optimum operational conditions like
pH, temperature etc. are very essential.
Within the normal range, changes in
temperature, pH and concentrations of
the substrate and enzyme affect the rate
of reaction, in accordance with predict-
able interactions between the enzyme
and the substrate molecules. Applica-
tion of commercial enzymes in food
processing, their functions and sources
are well documented in literature.
Enzymes have been used for the
pretreatment of the plant material prior
to the conventional method for extrac-
tion to get better yield and quality of
bioingredients. Recent studies em-
ploying enzyme pretreatment for the
extraction of flavour components from
various plant materials have shown
enhancement in aroma recovery. En-
zymes such as cellulases, hemi-cellu-
lases and pectinases, and a combination
of these have been used for the pretreat-
Special Report
Chemical Weekly October 1, 2013216
ment of plant materials. The major
action of cellulase and hemi-cellu-
lase are on cell walls. They act on
cell wall components, hydrolyse
them and increase the permeabil-
ity of the cell wall, thus resulting
in higher yield of the metabolites.
In the following pages, details
of enzyme assisted extraction of
materials like natural flavours &
oils, natural colour pigments and
miscellaneous natural compounds
are discussed.
Enzyme assisted extraction of
natural flavours and oils
Volatile oil from cumin seeds
Cumin (Cuminumcyminum sp.)
of the family Apiaceae has been used as
a spice since ancient times. It is a native
of Mediterranean countries, but now
popular in other countries like India,
Turkey, Syria, China, USA, Indonesia
and Iran. India is the world’s largest pro-
ducer and consumer of cumin and it is
mainly grown in Gujarat, Rajasthan and
Uttar Pradesh. Cumin finds extensive
use in foods, beverages, liquors, medi-
cines, toiletries and perfumery. Cumin
seed is used as a flavouring agent,
either as whole seed or as ground pow-
der. Based on the application, different
value-added products are also made
from cumin seed, such as oleoresin
and volatile oil. Cumin contains 3-4 %
volatile oil, which is extracted by steam
distillation or hydro distillation. Studies
conducted on the effect of various
enzymes on the extraction of volatile
oil of cumin seeds, showed that the oil
yield, after pre-treatment of cumin seeds
with cellulase, pectinase, protease and
viscozyme, was in the range 3.2-3.3%,
compared to 2.7% in a control sample.
The study demonstrated that enzymes
facilitated the extraction of cumin oil
with increase in oil yield, with little
change in either flavour profile or physi-
cochemical properties of the oil.
Volatile oil from ginger
Ginger (Zingiber officiale roscoe)
is one of the most widely used species
of the family Zingiberaceae. In ancient
times ginger was used for flavour-
ing, but more valued for its medicinal
properties and therefore a constituent
of many pharmaceutical preparations.
Ginger is a natural dietary component,
which has antioxidant and anti-carci-
nogenic properties. Apart from salted,
pickled and juicy products, ginger is
currently processed into ginger pow-
der, oleoresin and oil. Gingerols and
shogaols are the major bioactive con-
stituents and are responsible for the
anti-inflammatory, antitumor and anti-
oxidant activities of ginger. The aroma
and flavour of ginger is determined
by its volatile oil. Lixiao and Liu
YangFang (2009) studied the extrac-
tion process of ginger essential oil by
enzyme treatment. The ginger was
treated with cellulase and the treatment
time, temperature, pH and enzyme
dosage was optimised to influence the
percentage of the amount of essen-
tial oil during extraction. The amount
of essential oil extracted obviously
improves using the cellulase treat-
ment.
Volatile oil from garlic
Garlic (Allium sativum Lin.)
has a long tradition of use as a
food and as a medicinal plant.
It belongs to the genus Allium,
which comprises of approximately
600 known species distributed
over the whole northern hemi-
sphere. Garlic is evaluated for its
flavour, which is due to sulphur-
containing volatile oil. Garlic oil
is extracted by steam distillation
or hydro distillation. Sowbhagya
et al, (2008) studied affect of en-
zyme-assisted extraction on qua-
lity of garlic volatile oil and found
that application of enzyme prior to
steam distillation/hydro distilla-
tion resulted in a two-fold increase
in the yield of oil. The oil yield in
case of cellulase, pectinase, protease
and viscozyme pretreatment was in the
range of 0.39-0.51%, as against 0.28%
in a control sample by steam distilla-
tion, and in the range of 0.45-0.57% by
hydro distillation as against 0.31% in a
control sample.
Profiling of the garlic oil thus
obtained was carried out by gas chro-
matography-mass spectrometry (GC-
MS). Di-2-propenyl trisulphide (52%)
along with the corresponding di- and
tetra-sulphides (11% and 5%) consti-
tuted the major portion of the oil. The
other major flavour compounds iden-
tified were methyl-2-propenyl trisul-
phide (11.8%), vinyl dithiins (9.9%)
and dithianes (4.1%). The studies
demonstrate that enzymes facilitate the
extraction of garlic oil, resulting in an
increase in the yield of oil, with little
change either in flavour profile or physi-
cochemical properties of the oil.
Enzyme assisted liquefaction of
ginger rhizome for the production of
spray-dried products
A novel process for the production
of spray-dried ginger powder and paste-
like ginger condiments were developed
Special Report
217Chemical Weekly October 1, 2013
on pilot plant scale by Schweiggert et
al., (2007). The process includes the
operations of fine grinding, enzymatic
hydrolysis, finishing, pasteurisation
and spray-drying. Before scaling-up,
the enzymatic hydrolysis was optimised
on laboratory scale using D-optimal
design and analysed by response surface
methodology considering the individu-
al and interactive effects of temperature
(40-50ºC), pH (4.0-6.0), and enzyme
concentration (500-5,000 ppm) on the
reduction of viscosity of the ginger ho-
mogenate. In-process determination of
gingerols and shogaols demonstrated
that pungency is hardly influenced by
cell wall degrading enzymes, but sig-
nificantly affected by temperature and
pH. An enzyme mixture composed of
cellulolytic and pectinolytic activi-
ties at a 2:1 ratio yielded maximal tis-
sue digestion and highest retention of
pungent principles within two hours,
applying a dosage of 5,000-ppm at
40ºC and pH of 4.0. During processing
the amounts of 4-, 6-, 8- and 10-gin-
gerol slightly diminished, while 6 and
8-shogaol faintly increased. The ginger
digest obtained after finishing turned
out to be a valuable raw material to be
processed into various ginger products.
Pasteurisation and spray drying resulted
in homogenous paste-like ginger prepa-
rations and spray-dried ginger powder,
respectively. Additionally, the solid resi-
due contained large amounts of pungent
principles, which enables its application
as a flavouring agent. Consequently, the
process described in this study allows an
exhaustive utilisation of ginger rhizomes
for the production of various ginger ap-
plications.
Volatile oil from black pepper and
cardamom
This study investigated the effect of
enzyme pre-treatment on extraction of
active compounds from spices, namely,
black pepper and cardamom. A mix-
ture of enzymes, namely, Lumicellulae
(a mixture of cellulase, β-glucanase,
pectinase, and xylanase), was used for
the pre-treatment of black pepper and
cardamom. The pre-treatment of spices
with enzyme increased the yield of es-
sential oil. The GC and GC-MS evalua-
tion of the essential oil showed that the
major active components in spices, such
as, β-caryophyllene in black pepper and
α-terpenyl acetate in cardamom, mark-
edly increased from 15.03% to 25.58%
and 38.91% to 48.6%, respectively, on
enzyme treatment as compared with the
untreated control (Chandran, 2012).
Volatile oil from celery seeds
Celery (Apiumgraveolens Linn.)
seed is both a spice and a condiment.
It is grown commercially in the USA,
France and other parts of Europe.
Celery seed is used in flavouring and
seasoning. Volatile oil obtained from
celery seed is used in perfumes and
pharmaceutical industries. Normally
the volatile oil is isolated by steam dis-
tillation, hydro distillation and solvent
extraction. Enzyme assisted extrac-
tion is a novel step in this field. Sow-
bhagya et al (2009) studied the effect of
enzymes on extraction of volatiles
from celery seeds. The oil yield, after
cellulase, pectinase, protease and vis-
cozyme pretreatment, was in the range
2.2-2.3% as against 1.8% in a control
sample, by steam distillation. Profiling
of the celery oil thus obtained by GC-
MS showed that limonene – the major
terpene – increased from 63% to 83%
with enzyme treatment.
Essential oils from clove
Clove (Syzygiumaromaticum Linn)
is one of the most ancient and valu-
able spices of the Orient. The essential
oil of clove holds an important posi-
tion amongst essential oils. A typi-
cal steam distillation process for the
extraction of clove oil provides a 10.1%
yield. Recent studies have looked at
the use of enzymes such as pectinase,
amylase, lignocellulase and cellulase
on the powder of clove buds, prior to
extraction. The traditional methods of
physical and chemical extraction are
effective, but may affect the structure,
quality and yield of the phytochemicals
extracted. In current studies, enzymes
specific for action on the cell wall have
been used in the pre-treatment prior to
extraction, to enhance the quality and
yield of the phytochemicals extracted.
The results indicated that all the
enzymes gave more than 50% higher
yield than control sample in terms of
weight of extracted essential oil. A
mixture of the enzymes gave the high-
est yield of 17.82%. Gas chromato-
graphy results indicated that the essen-
tial oil extracted using amylase had a
maximum eugenol content of 70%, in
comparison with the eugenol content
(62-68%) in the essential oils extracted
using the rest of the enzymes. Antibac-
terial activity of all the extracts was
studied on methicillin-resistant staphy-
lococcus aureus (MRSA). The essential
oil extracted by using amylase-inhi-
bited MRSA showed a zone size of 40-
mm, whereas the essential oil extracted
by using lignocellulase showed a zone
size of 45-mm. The gas chromatogram
indicated the maximum number of peaks
in this extraction, which could be pro-
ducing a combined antibacterial effect
on the organism. The specific gravity
values of the essential oil extracted using
lignocellulase and amylase were 1.051
and 1.062, respectively, whereas the
control had a specific gravity of 1.015.
Bioactive compounds from ginger
The effect of application of α-
amylase, viscozyme, cellulase, prote-
ase and pectinase enzymes to ginger
on the oleoresin yield and 6-gingerol
content was investigated. Pre-treatment
of ginger with α-amylase or viscozyme
followed by extraction with acetone af-
forded higher yield of oleoresin (20% ±
0.5) and gingerol (12.2% ± 0.4), com-
pared to control (15% ± 0.6 of oleo-
resin, 6.4% ± 0.4 of gingerol). Extrac-
tion of ginger pre-treated with enzymes
Special Report
Chemical Weekly October 1, 2013218
followed by extraction with ethanol
provided higher yield of gingerol (6.2-
6.3%) than the control (5.5%) with
comparable yields of the oleoresin
(31-32%). Also, ethanol extraction of
cellulase pre-treated ginger had the
maximum polyphenol content (37.5
mg/g). Apart from 6-gingerol, 6-para-
dol along with 6- and 8-Me shogaols
were the other important bio-active
constituents in the oleoresin from cel-
lulase-treated ginger.
Enzyme-assisted extraction of
bioactives from plants
Demand for new and novel natural
compounds has intensified the deve-
lopment of plant-derived compounds
known as bioactives that either pro-
mote health or are toxic when ingested.
Enhanced release of these bioactives
from plant cells by cell disruption and
extraction through the cell wall can be
optimised using enzyme preparations
either alone or in mixtures. However,
the biotechnological application of
enzymes is not currently exploited to
its maximum potential within the food
industry.
Extraction of oil from Ricinoden-
dronheudelotii
Enzymatic pretreatment with a pro-
tease and cellulase in the Ricinoden-
dronheudelotii oil extraction process
were studied and this was carried out
by factorial experiment involving three
enzyme concentration and three treat-
ment times for each enzyme. Enzy-
matic hydrolysis significantly (p<0.05)
increased the oil extraction yield. The
highest improvement was obtained for
R.heudelotii seeds treated with Prota-
mex, which gave 15% (w/w) increase.
Extraction of oil and protein from
soybean
The effect of enzymes protease
and cellulase on oil & protein extrac-
tion yields of soya bean combined
with other process parameters such as
enzyme concentration, time of hydro-
lysis, particle size and solid-to-liquid
ratio was evaluated by response surface
methodology. The selection of enzyme
for the study was based on preliminary
experiment that showed higher incre-
ments in the extraction yield with the
use of two enzymes when compacted to
hemicellulase and pectinase. The use of
protease resulted in significantly higher
yields over the control, protein yield
increased from 27.8% to 66.2%, and oil
yield increased from 41.8% to 58.7%
only when heated flour was used, or
when non-heat treated flour with large
particle size was used in extraction.
Extraction of soya bean oil
An enzymatic treatment with carbo-
hydrases was performed either simul-
taneously with or prior to the hexane
extraction of oil from soya grits. The
enzymatic treatment increased the oil
extractability by 5% of the extractable
oil when it was carried out simultane-
ously with the oil extraction and 8-10%
if the treatment was carried out prior
to the solvent extraction. For this lat-
ter case the fraction easily extractable
increased up to 7.5%. Digestibility of
the meal was slightly improved by 3%
and the enzyme assisted extracted oil
contained higher free fatty acids and
phosphorus contents than the oil from
untreated samples.
Aqueous extraction of rice bran oil
Rice bran oil was extracted by
enzyme-assisted aqueous extraction
under optimised aqueous extraction
conditions using mixtures of protease,
amylase and cellulase. The optimal
conditions used yielded a 77% reco-
very of the oil.
Extraction of oil from soy and
sunflower seed
Enzymatic hydrolytic treatments to
enhance the oil extractability from soy
and sunflower seeds (low and high oil
content, respectively) were performed
at a range of experimental conditions
(moisture content, particle size, incu-
bation time, pH and agitation). Light
microscopy and scanning electron
microscopy showed the micro-structural
changes caused by the enzymatic attack.
The extent of the cell wall degradation,
closely related to the oil extractability,
was observed to be dependent on the
operational conditions under which the
enzymatic treatment was carried out.
Additionally, the effects of the enzymatic
treatment on the seed structure were
compared with those caused by thermal
and mechanical treatments.
Enzyme assisted extraction of
natural colour pigments
Lycopene from tomato processing
waste
Natural lycopene is produced by
extraction and concentration from whole
tomato fruits that are grown specifically
for this purpose. The commercially
available product, however, is very
expensive. This has prompted the search
for alternative sources of lycopene
and appropriate technologies for its
recovery. Antonio et al., (2011) studied
enzyme-assisted extraction of lycopene
from the peel fraction of tomato pro-
cessing waste. Overall, an 8- to 18-fold
increase in lycopene recovered was ob-
served compared to the untreated plant
material. The obtained results strongly
supports the idea of using cell wall
degrading enzyme as an effective means
for recovering lycopene from tomato
waste.
Extraction of turmeric oleoresin
Turmeric (Curcuma longa L.) is
one of the essential spices used as an
important culinary ingredient all over
Special Report
219Chemical Weekly October 1, 2013
the world as a colorant and a flavour-
ing agent. Turmeric contains curcumi-
noids that have antimutagenic and an-
tioxidant activities, and is the basis of
development of food formulations for
the prevention of cancer. Kurmudle et
al (2010) evaluated a novel technology
for turmeric extraction – enzyme assis-
ted three phase partitioning (EATPP), a
technique which has been explored for
protein separation. The process consists
of simultaneous addition of t-butanol
and ammonium sulphate to the aqueous
slurry of turmeric powder. The extrac-
tion of oleoresin was optimised with res-
pect to the concentration of ammonium
sulphate loading and the ratio of t-buta-
nol to slurry. Pretreatment of the slurry
with a commercial enzyme preparation
of α-amylase or glucoamylase followed
by three-phase partitioning was carried
out. The extraction time with this tech-
nique is lower as compared to conven-
tional acetone extraction.
EATPP is a new approach of ob-
taining efficient extraction of oleoresin
from turmeric. Unlike the convention-
al solvent extraction, which requires
12 hours for complete extraction, this
method takes only about four hours for
comparable yields of extractives. Al-
though EATPP is an efficient alterna-
tive to oleoresin extraction, extractive
scale-up studies are admittedly required
before one can recommend it for use at
the industrial level.
Lutein from marigold flowers
Marigold (Tageteserecta) is an
excellent and most important source of
carotenoids, the yellow carotenoids such
as carotenes (α- and β-carotinene) and
xanthophylls (lutein, zeaxanthin). These
find applications as an excellent antioxi-
dant for nutritional, cosmetic and phar-
maceutical industry. It is reported in the
literature that the risk of chronic disease,
such as heart disease, cancer and age-re-
lated eye diseases might be significantly
reduced by diets rich in lutein. Lutein is
a carotenoid found in dark green, leafy
vegetables such as spinach and various
fruits and flowers. The most important
source of lutein is flower petals of mari-
gold; here lutein is chemically bound
to various types of fatty acids such as
lauric, mystric and palmitic acids. Ex-
periments were conducted to study the
effect of non-commercial enzymes like
cellulase and pectinase on extraction of
lutein from marigold. The results have
demonstrated enhanced extraction yield
of 9.6% for lutein after treatment of the
dried and powdered flower petals with
enzymes and 5.2% without enzyme
addition. The isolated lutein was further
subjected to spectral analysis and quan-
tification by high performance liquid
chromatography.
Natural colorants from pomegranate
rind
The pomegranate (Punicagranatum)
is a naturally dense, deciduous, bushy,
multi-stemmed shrub and bears highly
coloured fruit with many juicy seeds
inside. The edible portion of the fruit,
called an aril, is comprised of hundreds
of seeds surrounded by juicy pigments,
each contained within a seed coat. Seeds
are either soft or hard, depending on the
cultivar. The juice within the aril varies
from light pink to dark red, but can also
appear yellow or clear in some varie-
ties. The juice ranges from very acidic to
very sweet in taste. The rind is generally
smooth but leathery, and can be yellow,
orange or red in colour. The rind is used
for extracting biopigments.
Ultrasound, enzyme and enzyme-
mediated ultrasound assisted extraction
processes have been used for the extrac-
tion of dye from pomegranate rind and
yields are found to be 29.2%, 26.5%,
35.6% and 8.8%, respectively. The
dye obtained has been used for dyeing
wool and cotton, keeping the optimum
dye bath concentration as 10% and 8%
(w/v) respectively. The fastness proper-
ties of wool are found to be very good,
while that of cotton is only satisfactory.
Enzyme assisted extraction of
miscellaneous natural compounds
Extraction of chlorogenic acid and
other phenols from spent coffee
Manuel et al., (2007) studied the
extraction of chlorogenic acid and other
phenols from spent coffee. Spent coffee
ground waste resulting from the industri-
al preparation of instant coffee was sub-
jected to solid-liquid extraction to study
the influence of variables like particle
size. The highest yields of phenols were
consistently obtained from the smaller
particles and an unexpected reduction in
phenol yields was consistently observed
from the smallest particles. An unex-
pected reduction in the phenol release
was observed when extraction was as-
sisted by cellulase treatment. Aqueous
ethanol (60%w/w) was the solvent hav-
ing the highest phenol-extractive capa-
city. High performance liquid chroma-
tography analysis confirmed chloro-
genicacidasthemajorphenolicacidbeing
extracted from the spent coffee grounds.
Chromatograms of extracts obtained
after the enzyme treatment showed that
cellulases catalysed the transformation
of chlorogenic acid.
Enzyme-assisted process for juice ex-
traction and clarification from litchis
Litchi pulp was treated with vari-
ous concentration levels of hydrolytic
enzymes viz. pectinase (0-0.133%
w/w), cellulase (0-0.266% w/w) and
hemicellulase (0-0.20% w/w) for dif-
ferent duration (30-150 mins) at 45°C.
Special Report
Chemical Weekly October 1, 2013220
The effect of enzyme treatment
conditions was studied on yield,
clarity, apparent viscosity and
total soluble solids (TSS) of
juice obtained from the pulp.
The optimum process condi-
tions were determined by em-
ploying a second order central
composite rotatable design in
combination with response sur-
face methodology. Yield, clarity
and TSS of juice were found to
increase and apparent viscosity
was found to decrease signifi-
cantly by enzymatic treatment.
Antioxidative phenols from black
current juice press residue
The enzymatic release of phenolic
compounds from pomace remaining
from black currant (Ribesnigrum) juice
production was examined. Treatment
with each of the commercial pectino-
lytic enzyme preparations – Grindamyl
pectinase, Macer8FJ, Macer8R and
Pectinex BE – as well as treatment
with Novozym89 protease significantly
increased plant cell wall breakdown
of pomace. Each of the tested enzyme
preparation except Grindamyl pectinase
also significantly enhanced the amount
of phenols extracted from pomace.
Macer8FJ and Macer8R decreased the
extraction yield of anthocyanins, where-
as Pectinex BE and Novozym89 protease
showed no effect. A decrease in pomace
particle size from 500-1,000-µm to less
than 125-µm increased the phenol yield
by 1.6-5 times.
Wet separation of starch from other
seed components of hull-less barley
Zheng and Bhatty (1998) studied
a multiple enzyme mixture contain-
ing cellulase, endo-(1-3), (1-4) β-D-
Glucanase and xylanase in wet separa-
tion of starch, protein, β-glucan, bran
and tailings from four hull-less barleys
(HB) viz., SB94794 (0% amylose),
CDC candle (5% amylose), CDC dawn
(24% amylose) and SBI550831 (40%
amylose). Compared to the convention-
al procedure it was found that the en-
zyme-assisted wet extraction reduced
slurry viscosity by 50-90%, the amount
of water and ethanol used in screening
and β-glucan precipitation by 30-60%
and screening time by 20-80%. The
enzyme-assisted extraction reduced
starch content and yield of tailings and
bran fractions, resulting in a 10% in-
crease in average starch extraction ef-
ficiency. However, β-glucan yield was
reduced in the enzyme-assisted extrac-
tion, particularly in high viscosity HB.
The physicochemical properties of the
isolated starches were not affected by
the enzyme assisted extraction.
Extraction of starch from sweet
potato
The effect of endogenous and com-
mercial hydrolytic enzyme (cytolase,
pectinase and cellulase) on improving
extraction of sweet potato starch was
studied. Starch granules present in sweet
potato roots are imbedded in cellulosic
fibres and held together by pectin sub-
strates. Extraction of starch is done by
rasping of the chopped roots. The ex-
periments revealed that the inherent or
endogenous enzyme had no substantial
effect on improving starch extraction.
The addition of commercial enzyme
was found to increase the extraction of
starch considerably even at lower levels
of rasping. It was found that cytolase en-
zyme at 0.1% (v/w) level, that is a com-
bination of pectinase and carbohy-
drases, was able to enhance starch
extraction more than two and half
fold that of control. This mixture
of cytolase and pectinase at 0.1%
level also gave more that 95%
starch recovery. It was found that
enzyme addition could serve as
good alternative to increased rasp-
ing, which could break down the
starch granules and fibres. With
drop in price of the enzyme this
could lead to cost saving over the
amount spent in rasping.
Extraction of silybin from the seeds of
Silybum marianum
Hong et al., (2009) studied optimi-
sation of enzyme assisted extraction of
silybin from the seeds of Silybum mari-
anum by Box-Behenken experimental
design. The important factors of the
enzyme-assisted extraction were op-
timised by employing Box-Behenken
design with the aid of the orthogonal
array design. The effect of enzyme with
respect to the incubation temperature,
the pH of enzyme solution and size
of the seed and the yield of the sily-
bin were visualised as three-dimension
response surface and contour plots. The
predictive yield was 24.6 mg/g defatted
seeds under the optimum enzymolysis
condition. The actual yield of silybin
was 24.81±1.93 mg/g defatted seeds,
higher by 138% and 123.6% than the
ethanol extraction in this study and in
the previous literature, respectively.
Extraction of antioxidants from
vegetable matrix
The enzyme assisted extraction of
antioxidants release of phenols from
vegetal matrixes and the enhanced
released of phenolic compounds at-
tached to the cell wall has been success-
fully addressed by means of an enzyme
treatment with degrading enzymes.
Commercial enzyme preparation of
pectinolytic, cellulolytic and proteoly-
tic activities, either alone or in mixture,
Special Report
221Chemical Weekly October 1, 2013
are suited for the purpose. Enhanced
extraction of phenolic compounds with
antioxidant activity from fruit juice and
wine has been extensively studied.
Extraction of black tea
Chandini et al (2011) investigated
improvement of the quality of black tea
extract with pre-treatment of pectinase
and tannase independently, successively
and simultaneously. Pectinase improved
the extractable solid yield (ESY) up to
11.5% without much of an improve-
ment in polyphenols recovery (14.3%).
Among the four treatments, tannase-
alone treatment showed the maximum
improvement in tea quality with higher
polyphenols in extracted solids. Treat-
ments involving tannase resulted in the
significant release of gallic acid. The
result suggested that employing a single
enzyme tannase for the pre-treatment of
black tea is desirable. Enzymatic extrac-
tion may be preferred over enzymatic
clarification, as it not only displayed
reduction in tea cream and turbidity,
but also improved the recovery of poly-
phenols and ESY in the extracts, as well
as maintaining a good balance of tea
quality.
Conclusion
In addition to the aforementioned
plants materials a number of other
plants like vanilla, pepper, mace, mus-
tard, fenugreek, rose and citrus peel,
which are potential source of bioingre-
dients have been studied for enzyme-
assisted extraction of spice oils and
oleoresins. Similarly, enzyme-assisted
extraction of colour has been studied in
plant materials like marigold, safflower,
grapes, paprika, tomato, alfalfa and
cherries. Efforts were made to develop
new technologies with enzyme pre-
treatment that could provide a thorough
extraction of flavouring principles from
vegetables.
Application of enzymes for bioin-
gredient extraction from plant mate-
rials is a new area, which requires more
intense research inputs to establish
itself as a promising technique. En-
zyme-assisted bioingredient extraction
has been worked upon for volatile oil
(cumin, garlic, celery, ginger), tomato
products (lycopene), chlorogenic acid
(green coffee), chilli (carotenoids),
marigold (lutein), starch (sweet potato),
strawberry and grapes (anthocyanin),
and alfalfa (chlorophyll) using en-
zyme preparations containing cellulase,
hemicellulase, pectinase, and glycosi-
dase. Enzyme pretreatment of cumin,
celery and garlic have resulted in en-
hanced oil recovery.
Application of enzymes for complete
extraction of bioingredients without the
use of solvents could be an attractive
proposal. Application of enzymes in
solvents could be tried for enhanced ex-
traction of value-added cell constituents
like major non-volatile component from
spices. The key advantages of enzyme
pretreatment include the reduction in ex-
traction time, minimal usage of solvents,
and a product with increased yield and
quality.Alimitation of this method could
be the cost of the enzymes. This could be
overcome by balancing the concentra-
tion of enzyme preparations and tailor-
made enzyme preparations for specific
reactions. In some commercial applica-
tions, crude enzyme preparations can be
used, which can reduce the cost of the
enzyme preparation. The increased yield
of value-added products (volatile oils)
obtained by the enzyme pre-treatments
can balance the increased cost of using
enzymes. Knowledge of the cell wall
compositions of the raw material to be
treated helps in the selection of the en-
zyme and the concentration to be used.
Enzymes have been used safely in a
wide variety of foods for centuries. The
biodiversity of enzymes is providing
the food industry with a wide range of
functionalities. In case of herbs, which
are quite sensitive to heat, the drying and
extraction of flavour from fresh herbs
is time consuming and cumbersome.
Enzymatic extraction offers a very good
choice of extraction of herbs, which
needstobeexploited.Newenzymeswith
added functionalities can be designed by
biotechnology. There is a wide scope for
further research for the preparation and
evaluation of tailor-made enzymes for
the extraction of value-added products
from plant materials not exploited so far.
Enzyme cocktails obtained by genetic
engineering can be highly efficient (with
Special Report
Chemical Weekly October 1, 2013222
enhanced functionality of the enzyme),
requiring low doses for the required ef-
fects, thus reducing the cost of enzymes
used in the process.
The prospects of enzyme media-
ted flavour and colour extraction from
plant materials appear to be very good
and promising in terms of future tech-
nology.
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Bioingredients article- First bublication

  • 2. Special Report 213Chemical Weekly October 1, 2013 Enzyme-assisted extraction of bioingredients I n the modern world, bioingredi- ents have an important role to play in different spheres of human life. They can be a major component of food we eat; beverages that quench our thirst; medicines that cure our illness; cosmetics that make us beautiful and clothes that make us fashionable. So the demand for bioingredients is increasing day by day. Modern science and tech- nology is being challenged by the rising demand from society for development of new bioingredients, sources and method of isolation. Bioingredients are the active prin- ciples of plants and are generated in the plants either by the primary or second- ary metabolic reactions taking place during the course of plant growth. The primarymetabolitesofplantsaremainly lipids, protein and carbohydrates. The secondary metabolites are classified into three main groups: phenolics, ter- penoids and alkaloids. As a consequence of the increased demand for bioingredients like essential oil, natural colours, pure components from the industries, there is a need for extensive collection of information on the composition of bioingredients and their recovery methods. The extraction of bioingredients from plant material can be achieved by a number of different methods, like: Expression; Distillation (hydro- or water-distil- lation, water and steam distillation, steam distillation); Solvent extraction; Super Critical Fluid Extraction (SCFE); and Enzyme assisted extraction. For each method there may be many variations and refinements and the extraction may be conducted under reduced pressure (vacuum), ambient pressure or excess pressure. The choice of extraction method will depend on the nature of the material, the stability of the chemical components and the speci- fication of the targeted product. Expression Expression is used exclusively for the extraction of citrus oil from the fruit peel, because the chemical components of the oil are easily damaged by heat. Citrus oil production is now a major by- product process of the juice industry. Distillation Distillation is a physical process used in most of the industries to iso- late the volatile portions of plant ma- terials. The main advantage of distilla- tion is that it can generally be carried out with some very simple equipment, close to the location of plant produc- tion. Even in relatively remote loca- tions large quantities of material can be processed in a relatively short time. There are various types of distillation methods like hydro distillation, water and steam distillation and steam distil- lation. The selection of the distillation method depends on the nature of the plants and their parts. Water distillation is the simplest of the three distillation methods. It is one of the oldest methods of distilla- tion used. The plant material is mixed directly with water in a still pot. A perforated grid may be inserted above the base of the still pot to prevent the plant material settling on the bottom and coming in direct contact with the heated base of the still and resulting in charring. The spice is fully immersed in hot water. The result is a soup, which carries aromatic molecules of the plant. The method is not much in use these days, because of the risk of overheating K.C. BABY Lead-Sales Application Bio-Ingredients Division Synthite Industries Ltd. E-mail: babykc@synthite.com DR. T.V. RANGANATHAN Professor Food Processing and Engineering Karunya University, Coimbatore E-mail: srivarahe@gmail.com
  • 3. Special Report Chemical Weekly October 1, 2013214 the plant and subsequent loss of the oil. The water-distilled oils are commonly darker in colour and have stronger still ‘off-note’ odours than oils produced by other methods, and therefore tend to be of the lowest value. Generally, this method is followed at the laboratory scale to screen the quality of the raw material. Hydro distillation is not commonly practiced in industries because of the long distillation time and the resulting mass obtained after hydro distillation is not easily amenable for oleoresin extraction with solvents. In steam-and-water distillation, the basic still design is very similar to that of water distillation. The plant material is packed into the still pot sitting on a grill or perforated plate above the boil- ing water. The capacity of the still pot volume is reduced, but it may be pos- sible to achieve a high packing density because the plant material is not sus- pended in the water. The advantages of steam and water distillation over water distillation include: Higher oil yield; Oil component less susceptible to change due to wetness and thermal conductivity of the still from the heat source; The effect of refluxing is mini- mised; Oil quality is more reproducible; and Faster process, so more energy efficient. Steam distillation is the process of distilling plant material with the steam generated outside the still in a stand- alone boiler. As in the steam-and-water distillation system the plant material is supported on a perforated grid above the steam inlet. The powdered raw material is charged into the still and steam is introduced from the bottom of the still. The steam carries off the vola- tile components and passes through a condenser where the volatiles get con- densed and separated from water. Most of the spices such as pepper, ginger, cardamom, etc. are subjected to steam distillation to obtain essential oils from the spice. Solvent extraction Many herbs and spices cannot be extracted by distillation method. In such cases, solvent extraction is the safest method for extracting high qua- lity oil. In this process, the spices or herbs are immersed in the solvent and the ‘separation’ is performed chemi- cally. These include pigments, volatile molecules and non-aromatic waxes. A solvent is a liquid or gas that dissolves a solid, liquid or gaseous solute, resul- ting in a solution. Solvents usually have a low boiling point and evaporate easily, or can be removed by distilla- tion, leaving the dissolved substance behind. Basically there are two cate- gories of solvent, i.e., organic and in- organic solvent. The selection of an appropriate solvent is guided by theory and experience. Generally, a good sol- vent should meet the following criteria: It should be inert to the reaction conditions; It should dissolve the reactants, reagents and bio actives; It should have an appropriate boil- ing point; and It should be easily removed at the end of the reaction. Spice oleoresins are prepared by this method. Super critical fluid extraction (SCFE) Solvent extraction is not considered the best method for extraction as the sol- vents can leave a small amount of resi- due behind which could cause allergies and affect the immune system. The most recent method is that of the distinguished carbon dioxide extraction. This method uses carbon dioxide to extract the essentialoilandoleoresinsfromtheplant when liquefied under pressure. Once the liquid depressurises, the carbon dioxide returns to a gaseous state, and only pure essential oil and resin remain. It employs a much lower temperature than that of steam distillation. This process is intend- ed to introduce oils that remain close to the way they reside in nature. This is pri- marily due to the inert nature of the sol- vent and lower pressures. There is some disagreement to this theory in that due to the acidic nature of CO2 one could argue it is disruptive to the chemistry of the resultant oil. Whereas terpenes are often manufactured through the methodology of distillation, lower terpene content is produced through a CO2 extraction, a higher ester range is manufactured, and the addition of molecules far too large to pass their way over through distillation can be found. These findings confirm that the oil does have more of the botan- ics inherent personality. SCFE has many advantages over other methods of extraction like the absence of solvent residues, no off- odours, low mono-terpenes hydrocar- bon levels, more top notes and more back notes giving a full but different profile than with traditional extraction methods.
  • 4. Special Report 215Chemical Weekly October 1, 2013 Enzyme assisted extraction Modern science and technology have helped flavour industries, food industries, cosmetics and other non-food industries to improve product qualities by providing advanced equipments and ingredients. To cop up with the emerg- ing requirements, advanced extraction technologies are required. Enzyme assisted extraction is the recent approach for extracting bioingredients from plant materials. The applications of enzymes in the extraction of essential oils from oilseeds like sunflower, soybean, rape- seed, corn, coconut, olives, avocado, and also for the extraction of rice bran oil etc. are well documented. Enzymes are proteins with highly specialised catalytic functions and pro- duced by all living organisms. They are responsible for many essential bio- chemical reactions in microorganisms, plants, animals and human beings. Enzymes are essential for all metabolic processes. Although like all other pro- teins, enzymes are composed of amino acids, they differ in function in that they have the unique ability to facilitate biochemical reactions without under- going change themselves. This cata- lytic capability is what makes enzymes unique. In other words, they are highly specific biological catalysts. Enzymes not only work efficiently and rapidly, they are also biodegradable. Enzymes are highly efficient in increasing the reaction rate of biochemical processes that otherwise proceed very slowly, or in some cases, not at all. Enzymes are categorised according to the compounds they act upon. Some of the most common types include pro- teases (which break down proteins), cel- lulases (which break down cellulose), lipases (which split fats into glycerol and fatty acids), and amylases (which break down starch into simple sugars). For example, the enzyme lipase exerts no action on starch substrate and amy- lases do not act on the proteinaceous substrate. This defines its ‘specificity’ and provides the basis of its classifica- tion and name. Often, the trivial name of the enzyme, derived from the trun- cated substrate name with ‘ase’ added, identifies the substrate or substrate range better for food technologists than does its systematic name or its Interna- tional Union of Biochemistry Enzyme Commission (IUB or EC) number. Enzymes are classified into the fol- lowing types: hydrolyzing enzymes, oxidation-reduction enzymes, ligases, group transfer enzymes, desmolases, isomerizing enzymes, and carboxy- lation enzymes. Based on their pro- perty of catalysing definite reactions, a particular enzyme acts on a specific substrate. The enzymes also do not become part of the final product of the bio- chemical reaction that they are catalys- ing. When the biochemical reaction is over, the product of the reaction leaves the enzyme. The enzyme is then ready to effect the same reaction on another molecule again and again. Given the right conditions to function, the enzyme can go on and on for as long as needed. Enzymes are large molecules with hundreds of amino acids. Only a small part of the enzyme participates in the catalysis of biochemical reactions; this is called the active site. The three- dimensional structure of the enzyme determines the appearance of the active site. The active site accommodates the shape of the biological substrate that requires transformation. They fit like a key in a lock. This is what makes enzymes specific in their actions. Only substances of the right shape will be transformed. The specificity of action of an enzyme on a specific substrate is determined by the structure and confor- mation of active site. Enzymes have been used for hun- dreds of years, and today the use of them is almost without limits. The his- torical uses of enzymes to make beer, wine, cheese and bread are elegant examples of the industrial exploitation of the power and selectivity of enzymes. In each case of enzyme application, one has to standardise the operational conditions viz., enzyme concentration and time of incubation, temperature of incubation, and optimum pH for the maximum activity of the enzyme. To use enzymes effectively in food appli- cations, proper knowledge about the nature of the enzyme, the source of the enzyme, active site, mode of action, optimum operational conditions like pH, temperature etc. are very essential. Within the normal range, changes in temperature, pH and concentrations of the substrate and enzyme affect the rate of reaction, in accordance with predict- able interactions between the enzyme and the substrate molecules. Applica- tion of commercial enzymes in food processing, their functions and sources are well documented in literature. Enzymes have been used for the pretreatment of the plant material prior to the conventional method for extrac- tion to get better yield and quality of bioingredients. Recent studies em- ploying enzyme pretreatment for the extraction of flavour components from various plant materials have shown enhancement in aroma recovery. En- zymes such as cellulases, hemi-cellu- lases and pectinases, and a combination of these have been used for the pretreat-
  • 5. Special Report Chemical Weekly October 1, 2013216 ment of plant materials. The major action of cellulase and hemi-cellu- lase are on cell walls. They act on cell wall components, hydrolyse them and increase the permeabil- ity of the cell wall, thus resulting in higher yield of the metabolites. In the following pages, details of enzyme assisted extraction of materials like natural flavours & oils, natural colour pigments and miscellaneous natural compounds are discussed. Enzyme assisted extraction of natural flavours and oils Volatile oil from cumin seeds Cumin (Cuminumcyminum sp.) of the family Apiaceae has been used as a spice since ancient times. It is a native of Mediterranean countries, but now popular in other countries like India, Turkey, Syria, China, USA, Indonesia and Iran. India is the world’s largest pro- ducer and consumer of cumin and it is mainly grown in Gujarat, Rajasthan and Uttar Pradesh. Cumin finds extensive use in foods, beverages, liquors, medi- cines, toiletries and perfumery. Cumin seed is used as a flavouring agent, either as whole seed or as ground pow- der. Based on the application, different value-added products are also made from cumin seed, such as oleoresin and volatile oil. Cumin contains 3-4 % volatile oil, which is extracted by steam distillation or hydro distillation. Studies conducted on the effect of various enzymes on the extraction of volatile oil of cumin seeds, showed that the oil yield, after pre-treatment of cumin seeds with cellulase, pectinase, protease and viscozyme, was in the range 3.2-3.3%, compared to 2.7% in a control sample. The study demonstrated that enzymes facilitated the extraction of cumin oil with increase in oil yield, with little change in either flavour profile or physi- cochemical properties of the oil. Volatile oil from ginger Ginger (Zingiber officiale roscoe) is one of the most widely used species of the family Zingiberaceae. In ancient times ginger was used for flavour- ing, but more valued for its medicinal properties and therefore a constituent of many pharmaceutical preparations. Ginger is a natural dietary component, which has antioxidant and anti-carci- nogenic properties. Apart from salted, pickled and juicy products, ginger is currently processed into ginger pow- der, oleoresin and oil. Gingerols and shogaols are the major bioactive con- stituents and are responsible for the anti-inflammatory, antitumor and anti- oxidant activities of ginger. The aroma and flavour of ginger is determined by its volatile oil. Lixiao and Liu YangFang (2009) studied the extrac- tion process of ginger essential oil by enzyme treatment. The ginger was treated with cellulase and the treatment time, temperature, pH and enzyme dosage was optimised to influence the percentage of the amount of essen- tial oil during extraction. The amount of essential oil extracted obviously improves using the cellulase treat- ment. Volatile oil from garlic Garlic (Allium sativum Lin.) has a long tradition of use as a food and as a medicinal plant. It belongs to the genus Allium, which comprises of approximately 600 known species distributed over the whole northern hemi- sphere. Garlic is evaluated for its flavour, which is due to sulphur- containing volatile oil. Garlic oil is extracted by steam distillation or hydro distillation. Sowbhagya et al, (2008) studied affect of en- zyme-assisted extraction on qua- lity of garlic volatile oil and found that application of enzyme prior to steam distillation/hydro distilla- tion resulted in a two-fold increase in the yield of oil. The oil yield in case of cellulase, pectinase, protease and viscozyme pretreatment was in the range of 0.39-0.51%, as against 0.28% in a control sample by steam distilla- tion, and in the range of 0.45-0.57% by hydro distillation as against 0.31% in a control sample. Profiling of the garlic oil thus obtained was carried out by gas chro- matography-mass spectrometry (GC- MS). Di-2-propenyl trisulphide (52%) along with the corresponding di- and tetra-sulphides (11% and 5%) consti- tuted the major portion of the oil. The other major flavour compounds iden- tified were methyl-2-propenyl trisul- phide (11.8%), vinyl dithiins (9.9%) and dithianes (4.1%). The studies demonstrate that enzymes facilitate the extraction of garlic oil, resulting in an increase in the yield of oil, with little change either in flavour profile or physi- cochemical properties of the oil. Enzyme assisted liquefaction of ginger rhizome for the production of spray-dried products A novel process for the production of spray-dried ginger powder and paste- like ginger condiments were developed
  • 6. Special Report 217Chemical Weekly October 1, 2013 on pilot plant scale by Schweiggert et al., (2007). The process includes the operations of fine grinding, enzymatic hydrolysis, finishing, pasteurisation and spray-drying. Before scaling-up, the enzymatic hydrolysis was optimised on laboratory scale using D-optimal design and analysed by response surface methodology considering the individu- al and interactive effects of temperature (40-50ºC), pH (4.0-6.0), and enzyme concentration (500-5,000 ppm) on the reduction of viscosity of the ginger ho- mogenate. In-process determination of gingerols and shogaols demonstrated that pungency is hardly influenced by cell wall degrading enzymes, but sig- nificantly affected by temperature and pH. An enzyme mixture composed of cellulolytic and pectinolytic activi- ties at a 2:1 ratio yielded maximal tis- sue digestion and highest retention of pungent principles within two hours, applying a dosage of 5,000-ppm at 40ºC and pH of 4.0. During processing the amounts of 4-, 6-, 8- and 10-gin- gerol slightly diminished, while 6 and 8-shogaol faintly increased. The ginger digest obtained after finishing turned out to be a valuable raw material to be processed into various ginger products. Pasteurisation and spray drying resulted in homogenous paste-like ginger prepa- rations and spray-dried ginger powder, respectively. Additionally, the solid resi- due contained large amounts of pungent principles, which enables its application as a flavouring agent. Consequently, the process described in this study allows an exhaustive utilisation of ginger rhizomes for the production of various ginger ap- plications. Volatile oil from black pepper and cardamom This study investigated the effect of enzyme pre-treatment on extraction of active compounds from spices, namely, black pepper and cardamom. A mix- ture of enzymes, namely, Lumicellulae (a mixture of cellulase, β-glucanase, pectinase, and xylanase), was used for the pre-treatment of black pepper and cardamom. The pre-treatment of spices with enzyme increased the yield of es- sential oil. The GC and GC-MS evalua- tion of the essential oil showed that the major active components in spices, such as, β-caryophyllene in black pepper and α-terpenyl acetate in cardamom, mark- edly increased from 15.03% to 25.58% and 38.91% to 48.6%, respectively, on enzyme treatment as compared with the untreated control (Chandran, 2012). Volatile oil from celery seeds Celery (Apiumgraveolens Linn.) seed is both a spice and a condiment. It is grown commercially in the USA, France and other parts of Europe. Celery seed is used in flavouring and seasoning. Volatile oil obtained from celery seed is used in perfumes and pharmaceutical industries. Normally the volatile oil is isolated by steam dis- tillation, hydro distillation and solvent extraction. Enzyme assisted extrac- tion is a novel step in this field. Sow- bhagya et al (2009) studied the effect of enzymes on extraction of volatiles from celery seeds. The oil yield, after cellulase, pectinase, protease and vis- cozyme pretreatment, was in the range 2.2-2.3% as against 1.8% in a control sample, by steam distillation. Profiling of the celery oil thus obtained by GC- MS showed that limonene – the major terpene – increased from 63% to 83% with enzyme treatment. Essential oils from clove Clove (Syzygiumaromaticum Linn) is one of the most ancient and valu- able spices of the Orient. The essential oil of clove holds an important posi- tion amongst essential oils. A typi- cal steam distillation process for the extraction of clove oil provides a 10.1% yield. Recent studies have looked at the use of enzymes such as pectinase, amylase, lignocellulase and cellulase on the powder of clove buds, prior to extraction. The traditional methods of physical and chemical extraction are effective, but may affect the structure, quality and yield of the phytochemicals extracted. In current studies, enzymes specific for action on the cell wall have been used in the pre-treatment prior to extraction, to enhance the quality and yield of the phytochemicals extracted. The results indicated that all the enzymes gave more than 50% higher yield than control sample in terms of weight of extracted essential oil. A mixture of the enzymes gave the high- est yield of 17.82%. Gas chromato- graphy results indicated that the essen- tial oil extracted using amylase had a maximum eugenol content of 70%, in comparison with the eugenol content (62-68%) in the essential oils extracted using the rest of the enzymes. Antibac- terial activity of all the extracts was studied on methicillin-resistant staphy- lococcus aureus (MRSA). The essential oil extracted by using amylase-inhi- bited MRSA showed a zone size of 40- mm, whereas the essential oil extracted by using lignocellulase showed a zone size of 45-mm. The gas chromatogram indicated the maximum number of peaks in this extraction, which could be pro- ducing a combined antibacterial effect on the organism. The specific gravity values of the essential oil extracted using lignocellulase and amylase were 1.051 and 1.062, respectively, whereas the control had a specific gravity of 1.015. Bioactive compounds from ginger The effect of application of α- amylase, viscozyme, cellulase, prote- ase and pectinase enzymes to ginger on the oleoresin yield and 6-gingerol content was investigated. Pre-treatment of ginger with α-amylase or viscozyme followed by extraction with acetone af- forded higher yield of oleoresin (20% ± 0.5) and gingerol (12.2% ± 0.4), com- pared to control (15% ± 0.6 of oleo- resin, 6.4% ± 0.4 of gingerol). Extrac- tion of ginger pre-treated with enzymes
  • 7. Special Report Chemical Weekly October 1, 2013218 followed by extraction with ethanol provided higher yield of gingerol (6.2- 6.3%) than the control (5.5%) with comparable yields of the oleoresin (31-32%). Also, ethanol extraction of cellulase pre-treated ginger had the maximum polyphenol content (37.5 mg/g). Apart from 6-gingerol, 6-para- dol along with 6- and 8-Me shogaols were the other important bio-active constituents in the oleoresin from cel- lulase-treated ginger. Enzyme-assisted extraction of bioactives from plants Demand for new and novel natural compounds has intensified the deve- lopment of plant-derived compounds known as bioactives that either pro- mote health or are toxic when ingested. Enhanced release of these bioactives from plant cells by cell disruption and extraction through the cell wall can be optimised using enzyme preparations either alone or in mixtures. However, the biotechnological application of enzymes is not currently exploited to its maximum potential within the food industry. Extraction of oil from Ricinoden- dronheudelotii Enzymatic pretreatment with a pro- tease and cellulase in the Ricinoden- dronheudelotii oil extraction process were studied and this was carried out by factorial experiment involving three enzyme concentration and three treat- ment times for each enzyme. Enzy- matic hydrolysis significantly (p<0.05) increased the oil extraction yield. The highest improvement was obtained for R.heudelotii seeds treated with Prota- mex, which gave 15% (w/w) increase. Extraction of oil and protein from soybean The effect of enzymes protease and cellulase on oil & protein extrac- tion yields of soya bean combined with other process parameters such as enzyme concentration, time of hydro- lysis, particle size and solid-to-liquid ratio was evaluated by response surface methodology. The selection of enzyme for the study was based on preliminary experiment that showed higher incre- ments in the extraction yield with the use of two enzymes when compacted to hemicellulase and pectinase. The use of protease resulted in significantly higher yields over the control, protein yield increased from 27.8% to 66.2%, and oil yield increased from 41.8% to 58.7% only when heated flour was used, or when non-heat treated flour with large particle size was used in extraction. Extraction of soya bean oil An enzymatic treatment with carbo- hydrases was performed either simul- taneously with or prior to the hexane extraction of oil from soya grits. The enzymatic treatment increased the oil extractability by 5% of the extractable oil when it was carried out simultane- ously with the oil extraction and 8-10% if the treatment was carried out prior to the solvent extraction. For this lat- ter case the fraction easily extractable increased up to 7.5%. Digestibility of the meal was slightly improved by 3% and the enzyme assisted extracted oil contained higher free fatty acids and phosphorus contents than the oil from untreated samples. Aqueous extraction of rice bran oil Rice bran oil was extracted by enzyme-assisted aqueous extraction under optimised aqueous extraction conditions using mixtures of protease, amylase and cellulase. The optimal conditions used yielded a 77% reco- very of the oil. Extraction of oil from soy and sunflower seed Enzymatic hydrolytic treatments to enhance the oil extractability from soy and sunflower seeds (low and high oil content, respectively) were performed at a range of experimental conditions (moisture content, particle size, incu- bation time, pH and agitation). Light microscopy and scanning electron microscopy showed the micro-structural changes caused by the enzymatic attack. The extent of the cell wall degradation, closely related to the oil extractability, was observed to be dependent on the operational conditions under which the enzymatic treatment was carried out. Additionally, the effects of the enzymatic treatment on the seed structure were compared with those caused by thermal and mechanical treatments. Enzyme assisted extraction of natural colour pigments Lycopene from tomato processing waste Natural lycopene is produced by extraction and concentration from whole tomato fruits that are grown specifically for this purpose. The commercially available product, however, is very expensive. This has prompted the search for alternative sources of lycopene and appropriate technologies for its recovery. Antonio et al., (2011) studied enzyme-assisted extraction of lycopene from the peel fraction of tomato pro- cessing waste. Overall, an 8- to 18-fold increase in lycopene recovered was ob- served compared to the untreated plant material. The obtained results strongly supports the idea of using cell wall degrading enzyme as an effective means for recovering lycopene from tomato waste. Extraction of turmeric oleoresin Turmeric (Curcuma longa L.) is one of the essential spices used as an important culinary ingredient all over
  • 8. Special Report 219Chemical Weekly October 1, 2013 the world as a colorant and a flavour- ing agent. Turmeric contains curcumi- noids that have antimutagenic and an- tioxidant activities, and is the basis of development of food formulations for the prevention of cancer. Kurmudle et al (2010) evaluated a novel technology for turmeric extraction – enzyme assis- ted three phase partitioning (EATPP), a technique which has been explored for protein separation. The process consists of simultaneous addition of t-butanol and ammonium sulphate to the aqueous slurry of turmeric powder. The extrac- tion of oleoresin was optimised with res- pect to the concentration of ammonium sulphate loading and the ratio of t-buta- nol to slurry. Pretreatment of the slurry with a commercial enzyme preparation of α-amylase or glucoamylase followed by three-phase partitioning was carried out. The extraction time with this tech- nique is lower as compared to conven- tional acetone extraction. EATPP is a new approach of ob- taining efficient extraction of oleoresin from turmeric. Unlike the convention- al solvent extraction, which requires 12 hours for complete extraction, this method takes only about four hours for comparable yields of extractives. Al- though EATPP is an efficient alterna- tive to oleoresin extraction, extractive scale-up studies are admittedly required before one can recommend it for use at the industrial level. Lutein from marigold flowers Marigold (Tageteserecta) is an excellent and most important source of carotenoids, the yellow carotenoids such as carotenes (α- and β-carotinene) and xanthophylls (lutein, zeaxanthin). These find applications as an excellent antioxi- dant for nutritional, cosmetic and phar- maceutical industry. It is reported in the literature that the risk of chronic disease, such as heart disease, cancer and age-re- lated eye diseases might be significantly reduced by diets rich in lutein. Lutein is a carotenoid found in dark green, leafy vegetables such as spinach and various fruits and flowers. The most important source of lutein is flower petals of mari- gold; here lutein is chemically bound to various types of fatty acids such as lauric, mystric and palmitic acids. Ex- periments were conducted to study the effect of non-commercial enzymes like cellulase and pectinase on extraction of lutein from marigold. The results have demonstrated enhanced extraction yield of 9.6% for lutein after treatment of the dried and powdered flower petals with enzymes and 5.2% without enzyme addition. The isolated lutein was further subjected to spectral analysis and quan- tification by high performance liquid chromatography. Natural colorants from pomegranate rind The pomegranate (Punicagranatum) is a naturally dense, deciduous, bushy, multi-stemmed shrub and bears highly coloured fruit with many juicy seeds inside. The edible portion of the fruit, called an aril, is comprised of hundreds of seeds surrounded by juicy pigments, each contained within a seed coat. Seeds are either soft or hard, depending on the cultivar. The juice within the aril varies from light pink to dark red, but can also appear yellow or clear in some varie- ties. The juice ranges from very acidic to very sweet in taste. The rind is generally smooth but leathery, and can be yellow, orange or red in colour. The rind is used for extracting biopigments. Ultrasound, enzyme and enzyme- mediated ultrasound assisted extraction processes have been used for the extrac- tion of dye from pomegranate rind and yields are found to be 29.2%, 26.5%, 35.6% and 8.8%, respectively. The dye obtained has been used for dyeing wool and cotton, keeping the optimum dye bath concentration as 10% and 8% (w/v) respectively. The fastness proper- ties of wool are found to be very good, while that of cotton is only satisfactory. Enzyme assisted extraction of miscellaneous natural compounds Extraction of chlorogenic acid and other phenols from spent coffee Manuel et al., (2007) studied the extraction of chlorogenic acid and other phenols from spent coffee. Spent coffee ground waste resulting from the industri- al preparation of instant coffee was sub- jected to solid-liquid extraction to study the influence of variables like particle size. The highest yields of phenols were consistently obtained from the smaller particles and an unexpected reduction in phenol yields was consistently observed from the smallest particles. An unex- pected reduction in the phenol release was observed when extraction was as- sisted by cellulase treatment. Aqueous ethanol (60%w/w) was the solvent hav- ing the highest phenol-extractive capa- city. High performance liquid chroma- tography analysis confirmed chloro- genicacidasthemajorphenolicacidbeing extracted from the spent coffee grounds. Chromatograms of extracts obtained after the enzyme treatment showed that cellulases catalysed the transformation of chlorogenic acid. Enzyme-assisted process for juice ex- traction and clarification from litchis Litchi pulp was treated with vari- ous concentration levels of hydrolytic enzymes viz. pectinase (0-0.133% w/w), cellulase (0-0.266% w/w) and hemicellulase (0-0.20% w/w) for dif- ferent duration (30-150 mins) at 45°C.
  • 9. Special Report Chemical Weekly October 1, 2013220 The effect of enzyme treatment conditions was studied on yield, clarity, apparent viscosity and total soluble solids (TSS) of juice obtained from the pulp. The optimum process condi- tions were determined by em- ploying a second order central composite rotatable design in combination with response sur- face methodology. Yield, clarity and TSS of juice were found to increase and apparent viscosity was found to decrease signifi- cantly by enzymatic treatment. Antioxidative phenols from black current juice press residue The enzymatic release of phenolic compounds from pomace remaining from black currant (Ribesnigrum) juice production was examined. Treatment with each of the commercial pectino- lytic enzyme preparations – Grindamyl pectinase, Macer8FJ, Macer8R and Pectinex BE – as well as treatment with Novozym89 protease significantly increased plant cell wall breakdown of pomace. Each of the tested enzyme preparation except Grindamyl pectinase also significantly enhanced the amount of phenols extracted from pomace. Macer8FJ and Macer8R decreased the extraction yield of anthocyanins, where- as Pectinex BE and Novozym89 protease showed no effect. A decrease in pomace particle size from 500-1,000-µm to less than 125-µm increased the phenol yield by 1.6-5 times. Wet separation of starch from other seed components of hull-less barley Zheng and Bhatty (1998) studied a multiple enzyme mixture contain- ing cellulase, endo-(1-3), (1-4) β-D- Glucanase and xylanase in wet separa- tion of starch, protein, β-glucan, bran and tailings from four hull-less barleys (HB) viz., SB94794 (0% amylose), CDC candle (5% amylose), CDC dawn (24% amylose) and SBI550831 (40% amylose). Compared to the convention- al procedure it was found that the en- zyme-assisted wet extraction reduced slurry viscosity by 50-90%, the amount of water and ethanol used in screening and β-glucan precipitation by 30-60% and screening time by 20-80%. The enzyme-assisted extraction reduced starch content and yield of tailings and bran fractions, resulting in a 10% in- crease in average starch extraction ef- ficiency. However, β-glucan yield was reduced in the enzyme-assisted extrac- tion, particularly in high viscosity HB. The physicochemical properties of the isolated starches were not affected by the enzyme assisted extraction. Extraction of starch from sweet potato The effect of endogenous and com- mercial hydrolytic enzyme (cytolase, pectinase and cellulase) on improving extraction of sweet potato starch was studied. Starch granules present in sweet potato roots are imbedded in cellulosic fibres and held together by pectin sub- strates. Extraction of starch is done by rasping of the chopped roots. The ex- periments revealed that the inherent or endogenous enzyme had no substantial effect on improving starch extraction. The addition of commercial enzyme was found to increase the extraction of starch considerably even at lower levels of rasping. It was found that cytolase en- zyme at 0.1% (v/w) level, that is a com- bination of pectinase and carbohy- drases, was able to enhance starch extraction more than two and half fold that of control. This mixture of cytolase and pectinase at 0.1% level also gave more that 95% starch recovery. It was found that enzyme addition could serve as good alternative to increased rasp- ing, which could break down the starch granules and fibres. With drop in price of the enzyme this could lead to cost saving over the amount spent in rasping. Extraction of silybin from the seeds of Silybum marianum Hong et al., (2009) studied optimi- sation of enzyme assisted extraction of silybin from the seeds of Silybum mari- anum by Box-Behenken experimental design. The important factors of the enzyme-assisted extraction were op- timised by employing Box-Behenken design with the aid of the orthogonal array design. The effect of enzyme with respect to the incubation temperature, the pH of enzyme solution and size of the seed and the yield of the sily- bin were visualised as three-dimension response surface and contour plots. The predictive yield was 24.6 mg/g defatted seeds under the optimum enzymolysis condition. The actual yield of silybin was 24.81±1.93 mg/g defatted seeds, higher by 138% and 123.6% than the ethanol extraction in this study and in the previous literature, respectively. Extraction of antioxidants from vegetable matrix The enzyme assisted extraction of antioxidants release of phenols from vegetal matrixes and the enhanced released of phenolic compounds at- tached to the cell wall has been success- fully addressed by means of an enzyme treatment with degrading enzymes. Commercial enzyme preparation of pectinolytic, cellulolytic and proteoly- tic activities, either alone or in mixture,
  • 10. Special Report 221Chemical Weekly October 1, 2013 are suited for the purpose. Enhanced extraction of phenolic compounds with antioxidant activity from fruit juice and wine has been extensively studied. Extraction of black tea Chandini et al (2011) investigated improvement of the quality of black tea extract with pre-treatment of pectinase and tannase independently, successively and simultaneously. Pectinase improved the extractable solid yield (ESY) up to 11.5% without much of an improve- ment in polyphenols recovery (14.3%). Among the four treatments, tannase- alone treatment showed the maximum improvement in tea quality with higher polyphenols in extracted solids. Treat- ments involving tannase resulted in the significant release of gallic acid. The result suggested that employing a single enzyme tannase for the pre-treatment of black tea is desirable. Enzymatic extrac- tion may be preferred over enzymatic clarification, as it not only displayed reduction in tea cream and turbidity, but also improved the recovery of poly- phenols and ESY in the extracts, as well as maintaining a good balance of tea quality. Conclusion In addition to the aforementioned plants materials a number of other plants like vanilla, pepper, mace, mus- tard, fenugreek, rose and citrus peel, which are potential source of bioingre- dients have been studied for enzyme- assisted extraction of spice oils and oleoresins. Similarly, enzyme-assisted extraction of colour has been studied in plant materials like marigold, safflower, grapes, paprika, tomato, alfalfa and cherries. Efforts were made to develop new technologies with enzyme pre- treatment that could provide a thorough extraction of flavouring principles from vegetables. Application of enzymes for bioin- gredient extraction from plant mate- rials is a new area, which requires more intense research inputs to establish itself as a promising technique. En- zyme-assisted bioingredient extraction has been worked upon for volatile oil (cumin, garlic, celery, ginger), tomato products (lycopene), chlorogenic acid (green coffee), chilli (carotenoids), marigold (lutein), starch (sweet potato), strawberry and grapes (anthocyanin), and alfalfa (chlorophyll) using en- zyme preparations containing cellulase, hemicellulase, pectinase, and glycosi- dase. Enzyme pretreatment of cumin, celery and garlic have resulted in en- hanced oil recovery. Application of enzymes for complete extraction of bioingredients without the use of solvents could be an attractive proposal. Application of enzymes in solvents could be tried for enhanced ex- traction of value-added cell constituents like major non-volatile component from spices. The key advantages of enzyme pretreatment include the reduction in ex- traction time, minimal usage of solvents, and a product with increased yield and quality.Alimitation of this method could be the cost of the enzymes. This could be overcome by balancing the concentra- tion of enzyme preparations and tailor- made enzyme preparations for specific reactions. In some commercial applica- tions, crude enzyme preparations can be used, which can reduce the cost of the enzyme preparation. The increased yield of value-added products (volatile oils) obtained by the enzyme pre-treatments can balance the increased cost of using enzymes. Knowledge of the cell wall compositions of the raw material to be treated helps in the selection of the en- zyme and the concentration to be used. Enzymes have been used safely in a wide variety of foods for centuries. The biodiversity of enzymes is providing the food industry with a wide range of functionalities. In case of herbs, which are quite sensitive to heat, the drying and extraction of flavour from fresh herbs is time consuming and cumbersome. Enzymatic extraction offers a very good choice of extraction of herbs, which needstobeexploited.Newenzymeswith added functionalities can be designed by biotechnology. There is a wide scope for further research for the preparation and evaluation of tailor-made enzymes for the extraction of value-added products from plant materials not exploited so far. Enzyme cocktails obtained by genetic engineering can be highly efficient (with
  • 11. Special Report Chemical Weekly October 1, 2013222 enhanced functionality of the enzyme), requiring low doses for the required ef- fects, thus reducing the cost of enzymes used in the process. The prospects of enzyme media- ted flavour and colour extraction from plant materials appear to be very good and promising in terms of future tech- nology. References ASTA. (1985). Official Analytical Methods of American Spice Trade Association, 4th Edition, Englewood Cliffs, New Jersey. Amudan, R.; Kamat, D. V.; Kamat, S. D. South Asian Journal of Experi- mental Biology (2011), 1(6), 248- 254. Anne. KatrineLandbo and Anne S. Meyer 2001. Enzyme Assisted Ex- traction of Antioxidative Phenols from Black Currant Juice Press Residues (Ribesnigrum) J.Agri.Food Chem.2001, 49, 3160-3177. Antonio Z., M Fidaleo and R Lavecchia. 2011. Enzyme Assisted extraction of lycopene from tomato processing waste. Enzyme and Mi- crobial Technology 49(2011)567-57. Blank, I., Jaeger, D., Zurbriggen and Beat, D. (2000). Flavourant prepared from fenugreek (Trigonel- lafoenumgraecum) seeds US Patent 6,013,289. Balasubramaniyan, D., Bryce, C. F. A., Green, J. and Jayaraman, K. (1996). Concepts in Biotechnology. Universities Press (India) Ltd. Barzana, E., Rubio, D., Santamaria, R. I., Garcia, Correa. Garcial, F., RidauraSaaz, V. E. and Lopez, M. A. (2002). Enzyme mediated solvent extraction of carotenoids from mari- gold flower (TageteserectaL), J. Ag- ric. Food Chem. 50(16):4491-4496. Borges. P. & Pino. J. (1993) The isolation of volatile oil from cum- in seeds by steam distillation. Die Nahurung, 37.123-126. Burnerie, P. M. (1998). Process of the production of natural vanilla extracts by enzymatic processing of green vanilla pods and extract thereby ob- tained. US Patent 5,705,205. Chandran, Janu;Amma, Keezheveet- til Parukutty Padmakumari; Menon, Nirmala; Purushothaman, Jaya- murthy; Nisha, Prakasan Food Sci- ence and Biotechnology (2012), 21(6), 1611-1617. Cinar I. (2005). Effect of cellulase and pectinase concentration on the colour yield of enzyme extracted plant carotenoids. Process Bio- chemistry40 (2):945-949. Coll, L., D. Saura., Ruiz, M. P. and Canovas, J. A. (1995). Viscometric control in the enzymatic extraction of citrus peel oils. Food Control. 6 (3):143-146. Delgado-Vargas, F. and Pardes-Lo- pez., O. (1997). Effects of enzymatic treatments on carotenoid extraction from Marigold flowers, (Tagete- serecta). Food Chem.58 (3):255-258. Delgado-Vergas, F. and Pardes- Lopez, G. (2002). Enzyme media- ted solvent extraction of carotenoids from marigold flower (Tageteserec- ta). Agric. Food Chem. 50(16):4491- 4496. Dominquez, H., Nunez, M. J. and Loma, Y. M. (1993). Oil extract- ability from enzymatically treated soybean and sunflower, range of operational variables. Food Chem. 46:277-284. Dominquez, H., Nunez, M. J. and Loma, Y. M. (1994). Enzymatic treatment to enhance oil extraction from fruits and oil seeds: a review. Food Chem. 49:271-286. Dominquez, H., Nunez, M. J. and Loma, J. M. (1995). Enzyme assisted hexane extraction of soyabean oil. Food Chem. 54(2):223-231. Enzyme Development Corporation (212) 736-1580 21 Penn Plaza New York. NY1001. A Buyer’s Guide to Enzymes. Enzyme Technical Association 1800 Massachusetts Avenue, N. W. Se- cond Floor Washington, DC 20036 “Enzymes A primer on use and bene- fits today and tomorrow” Enzyme Technical Association Oc- tober 2001.”Enzymes Questions and Answers”. Govindarajan, V.S. (1982). Ginger- Chemistry, technology and quality evaluation: Part 1.Critical Reviews in Food Science and Nutrition, 17,1- 257. Greco L., R. Riccio, S. Bergero,A.A. M. Del Re and M. Trevisan 2007. To- tal reducing capacity of fresh sweet peppers and five different Italian pep- per recipes. Food Chem., 103: 1127- 1133. Godfrey. T. and West. S. (eds) (1996) Industrial Enzymology, 609pp. Lon- don: Macmillan. Handelman GJ. The evolving role of carotenoids in human biochemistry. Nutrion 2001;17:818-822. H. Domingguez, M. J. Nunez and J. M. Lema. Enzyme Assisted hexane extraction of soya bean oil, Food Chemistry 54 (1995) 223-231. Health, H.B., G.A. Reineccius, Fla- vour Chemistry and Technology, Van Nostrand Reinhold, New York,1986, p.442. Hinneburg I., H.J. Damien, R. Hil- tunen. 2006. Antioxidant activities of extracts from selected culinary herbs and spices. Food Chemistry, 97: 122-129. Hong Liu, Xueling Du, Qipeng Yuan and Lei Zhu. Optimization of En- zyme Assisted Extraction of Silybin from the Seeds of Silybum Mari- anum by Box-Behnken Experimen- tal Design. Phytochemical Analysis 2009, 20, 475-483. Kaefer C.M. and J.A. Milner. 2008. The role of herbs and spices in can- cer prevention. Journal of Nutritional Biochemistry, 19: 347-361. Kamonrat R., L. Shank and G. Chai- rote. 2010. Phenolic content and
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