Enzymatic

Pretreatment on
Phytoconstituents
• Enzymes, besides playing a vital role in all living
organism also have vast number of industrial
applications.
•Scientists are busy exploring newer application
for enzymes. One such area where enzymes
being considered is improving the yield in herbal
drug extraction.
Herbal Drug Extraction
• Herbal plants are nature's gift to mankind for
improving and maintaining lifestyle
• There is need to know which constituents in
the medicinal herb are responsible for
therapeutic use therefore the need arises to
extract, isolate and characterize
phytoconstituents responsible for its
therapeutic use.
Traditional Methods of
Extraction
• Decoction
• Strong decoction
• Tincture
• Maceration
• Distillation/Extraction
• Cold Percolation
• Agitation/Distillation
• Solvent extraction
 Decoction
• Decoctions are usually the
method of choice when
working with tougher and
more fibrous plants, barks
and roots (and which have
water soluble chemicals).
• Standard dosages for
decoction are generally one-
half to one cup, two or three
times daily. Again, the
entire day's dosage can be
prepared in the morning (2-
3 cups at one time), and the
remainder refrigerated until
ready to use later in the day.
 Strong Decoction
• Depending on the type of plant material used,
strong decoctions are prepared in two general
ways.
• When straining, again, make sure to press on
the cut herb pieces in the strainer to get as
much moisture/decoction out of the herb
pieces.
 Tincture
• A tincture is an alcohol and water extract which is used when
plants have active chemicals that are not very soluble in water,
and/or when a larger quantity is prepared for convenience and
wanted for longer term storage. Many properly prepared plant
tinctures can last several years or more without losing potency.
The percentage of alcohol usually helps determine it's shelf-life:
the more alcohol used, the longer the shelf life.
• The type of alcohol can vary from vodka, rum, or 90 to 180 proof
grain alcohol (sold as "everclear" in liquor stores and sometimes
cheaper than vodka). Vodka is fine, but remember if it says 40
proof; it is 40% alcohol and the rest is water.
• Since this method uses a higher ratio of plant to liquid and helps
concentrate the chemicals through the use of alcohol, dosages
needed for tinctures are usually much less than infusions and
decoctions.
• The tincture can be placed directly in the mouth for immediate
absorption, or placed in a small amount of water or juice.
 Maceration
• This method of
preparation is certainly
the easiest.
• Normally this is used
for very tender plants
and/or fresh plants, or
those with delicate
chemicals that might be
harmed by heating or
which might be
degraded in strong
alcohol.
 Distillation/Extraction
• This is the original extraction
method. Technically,
distillation devices are
"modified soxhlet extracters."
• Our most sophisticated units
which use this process are also
vacuum compatible. Applying
a vacuum to this inside of the
main flask lowers the boiling
point of the solvent enabling
the operator to distill solvent
at much lower temperatures
and to distill much more
quickly.
 Cold Percolation
This is a traditional method of extraction
used by herbalists throughout the
world and it's very simple. Above a
flask or vessel is suspended a cone or
tube. The bottom of the tube has a
perforated base which holds ground
herb in place. Solvent is poured into
the top of the tube where it soaks
through the herb leaching out the
extract and then falling out the bottom
end of the tube into the flask. If
desired, the percolation tube can be
wrapped in heating tape to help
facilitate the extraction.
 Agitation/Distillation
• This is the accepted method used by most large,
industrial operations which are in the business of
processing herbal extracts. The main reason for
this is that the distillation/extraction method
outlined earlier becomes cost prohibitive and
very time consuming when sized up to industrial
scale.
• To completely leach out the plant material, more
solvent must be introduced into the agitation
vessel whereby the process is repeated. This
process may be repeated 2-4 times to completely
leach out the extract from your herb with each
successive run yielding a leaner batch of extract.
• A press or centrifuge is used to squeeze the liquid
out of the spent plant material.
 Solvent extraction
However there is a general
observation that with conventional
solvent extraction process the yield
of extractive seldom goes beyond
60-70 % in the first run. There fore
to recover the reminder repititive
extraction need to be done, which
makes the whole process
uneconomical. Moreover the
extraction with the solvent is the
energy demanding process,which
increarses the cost of operation.
 Advanced methods of
herbal extraction
• High Pressure- Supercritical/Sub critical
Extraction

• Enzymatic extraction
 High Pressure-
Supercritical/Sub critical
Extraction
• This is the most technologically
advanced extraction system in the
world. Research into the techniques and
applications of this amazing process is
ongoing and Eden Labs is at the
forefront of these investigations.
• The advantages of SFE are the
versatility it offers in pinpointing the
constituents you want to extract from a
given material and the fact that your
end product has virtually no solvent
residues left in it. (CO2 evaporates
completely) The downside is that this
technology is quite expensive.
 Enzymatic extraction
Enzyme alone or in combination with other enzyme (blend) can be
used in phytoextraction depending on the composition of the
substrate.
• Structure of plant cell wall
The structure of the plant cell wall is quite complex, and is made up
of various strata (the middle lamella, the primary wall, the
secondary wall, and the tonoplast). The wall of the mature cell is an
amorphous,isotropic, colloidal layer composed mainly of pectic
substances , the ‘primary wall’, or original cambial wall, an
anisotropic layer composed of cellulose and pectic substances, the
‘secondary wall’ deposited upon the primary wall and composed
largely of cellulose. The strata are generaaly composed of
compounds such as pectic substances, hemicellulose, cellulose, and
lipids, interwined with peripheral and integral protein.
• Extensive search has proven that specific enzymes for specific
cementing materials help to break down the hard structure by
increasing the permeability of cell wall and thus allowing the release
of bioactive substances. Also, the herbal extract quality depends on
the age of herbal species, temperature of extraction and the extent of
pulverization of herbs.
 Drawbacks of Advantages of enzymatic treatment over
Traditional other methods of extraction
Extraction
• High temperature during • Enzymatic treatment for extraction is different from
water extraction destroys solvent extraction since enzymes are biospecific and
the important heat labile attack only on their sustrates.
active ingredients present • Due to the specificity of enzymes, there is less
in the herbal extract.
chance of degradation of the phytoconstituent and
• All the bioactive also small quantities of enzyme can act on large
ingredients present within quantities of substrate.
the cell walls are not able • Enzymatic action damages cell wall, favouring
to get extracted to their
permeability of phytoconstituent especially the
optimum level due to
constituent of interest.
hard cement bounding
present within the cell • The profitable effects of utilizing enzymes have long
wall. been recognized, with regard in the increasing yields
• of the main products by reducing side products and
The color, flavor, aroma,
low waste treatment.
and consistency of the
final herbal extract are
never consistent from
batch to batch.
• The extraction using • Another advantage is the tailoring
solvents are generally of enzyming complexes to fit the
carried out at high processing requirements because of
temperatures,which may be the mild conditions that avoid
little or of no use if the drastic operational conditions.also
compound of interest is not use of enzyme is circumvented; as
thermostable.therefore, small quantities are required for
enzymatic pretreatment.also use of
enzyme may result in substantial
release of the substance of interest.
• Lower operation temperature can
be used in enzymatic extraction
minimizing energy requirements at
the same time suitable for
thermolabile compounds.usuing
enzyme for facilitated release will
increase the solvent efficiency, as
less solvent will be required for
complete extraction
 Enzymes
• What are enzymes?

• Properties of enzyme:

5. Enzyme preparations may consist of whole cells, parts of

cells, or cell-free extracts from the source used.
6. Active components have known molecular weights that range
from 12,000 to several hundred thousand.
7. Enzymes may be in liquid, semi-liquid, or dry form. An
enzyme in general and pectinase in particular is readily
soluble in water.
8. Enzymes are practically insoluble in alcohol, in chloroform,
and in ether. The liquids are generally in aqueous solution,
having many of the same properties of water, with the liquid
form boiling point slightly above 100° (212° F).
1. Dry preparations are off-white to tan amorphous,
finely divided powders.
2. Liquids usually range in color from tan to dark-
brown. Individual preparations are generally
characterized by functionality and activity rather
than the properties of the product.
 Enzymes used in Herbal Drug
Extraction
• Amylase
• Cellulase
• Papain
• Lipase
• Pectinlyase
• Combinations (blends of enzyme used in extraction)
A blend of protease and amylase used for the simultaneous
saccharification and fermentation of grain starch.
Product Application
CelluSEB-TL Cellulase used for
eetraction, for liquifaction
of plant materials and for
downstream processing
SEBstar-HTL Heat-stable alpha-amylase
used for high temp.
liquefaction of starch
containing grain mashes
Benefits
· Increases liquid or solid plant
extract yields by 5 - 20%
· Aqueous extraction at mild
temperatures and pH conditions
· Increases extract purity
· Increases extraction of cereal
grain starch by 2 – 5%
· Easier and more rapid solids
separation and concentration
· Trouble-free clarification and
filtration
· Increased processing capacity and
profitability
Excellent thermal stability for
liquefaction of steam jet cooked
starch
· Produces low-viscosity, dextrose
syrups in 90 min. at 80-90o
· Whole corn or grain liquefaction
at pH 5.8 and 80-85o
· Increases wort yield and grain
adjunct cooking capacity
SEBamyl-GL Glucoamylase used for
saccharification of whole
· Excellent thermal and pH
stability
grain starch to glucose · Produces high-DE, glucose
syrups in less than 48 hours
· Allows whole-corn or grain
starch saccharification to occur at
pH 4.8 and 65o
SEB-Neutral P Protease used for
conversion of grain
· Hydrolyzes both animal and
SEB-Neutral PL vegetable proteins
proteins to increase free-
· Breaks down and increase
amino nitrogen
protein dispersibility, solubility,
palatabilitiy and digestability

SEBferm-L A blend of amylases and · Improved yeast growth and

proteases used for the faster fermentation rate
simultaneous · Higher alcohol yields and DDG
saccharification and protein value
fermentation of grain · Reduced thin stillage viscosity
starch. and improved evaporation
· Whole-corn or grain starch
saccharification at pH 4.8 and
65oC
 Industrial applications of
enzymatic pretreatment.
• Reduced production cost
• More concentrated product/ less impurity in the final product
• Bio – transformation
• Examples on herb extraction by enzyme in Novozymes
• Research On Enzymatic Herbal Extraction
• Enzymatic extraction of coconut oil Yield improvements in the
extraction
• Enzymatic extraction of coconut oil using cellulase, alpha-
amylase, polygalacturonase, and protease(Enzyme blend)
• Enzymatic pretreatment on jojoba seeds to facilitate oil extraction
• Application of enzymatic pretreatment in bioprocessing
• Enzyme Applications For Agro-Processing in Developing
Countries: An Inventory of Current and Potential Applications
• The use of enzymes in fruit juice and cider processing
 Yield improvements in the
extraction

• Facilitate release of principle molecules by

degrading plant material to a more open and
loose structure
• Better release of water/solvent from press cake
by reducing water - retaining capacity. The
bigger run - off volume is increasing the yield.
Reduced Production Cost
• Less number of extraction is needed to get a good
total yield, when the first extraction is more efficient
with the help of cell wall degrading enzyme.
• Reduce need for wash out of insoluble
(precipitate/press cake) when water- retaining
capacity is reduced.
• Reduced viscosity in concentration/evaporation, save
time and energy, with the help of enzyme for
reduction on water retaining capacity.
More concentrated product/
less impurity in the final
product
• Some Herb Tonic has a problem with
haze/precipitate formation during storage. The
haze/ precipitate is often a very complex mix
of pectin - polyphenol - protein.
• The pectin and protein can be degraded during
the production process to small molecules that
cannot form haze and stable Tonic can be
produced.
 Bio - transformation
• Cleave the beta - glycoside bond to the sugar
side chain on the principal molecule by added
enzyme
• Removing sugar side chain form the principal
molecules, will in much case change the
biological activity of the molecule, to a more
effective drug. This is traditional been done by
acid hydrolysis or " natural fermentation"
 Examples on herb
extraction by enzyme in
Novozymes
• Phenolic anti-oxidants extraction can be
improved by enzymatic treatment

• Examples on soy bean isoflavone extraction

and biotransformation
 Use of ExtractSEB in Herbal
Extraction
ExtractSEB. ExtractSEB is a GRAS (generally recognized as
safe)enzyme formulation for use as aid for breaking down the
cellular cementing substances present in root, stem and leaves for
extracting alkaloids, glucosides, oleoresins and spices from the
herbal mass to achieve 100% extraction efficiency and quality
product.

• ExtractSEB acts efficiently over a wide temperature range of 30°C to 50°c

and at natural Ph thus preserving the important heat labile constituents of the
herb.
• 15% to 20% increase in alkaloids content has been confirmed by field trials.
• ExtractSEB helps to increase the total solid content in the herbal extract by
10 to 15%.
• ExtractSEB is non-corrosive, noninflammable and nonirritant and therefore
harmless and easy to handle.
• Color, flavor, aroma and consistency of the final herbal extract are consistent
from one batch to another.
 Enzymatic extraction of coconut
oil using cellulase, alpha-amylase,
polygalacturonase, and protease
(Enzyme blend)
• The use of enzymes to extract coconut oil has
produced higher yields than any of the traditional
methods, with a 12-30% increase
• The optimum oil recovery was with mixed enzymes,
yielding 73.8% at pH of 7 and 60 C.
• The enzymes used were cellulase, alpha-amylase,
polygalacturonase, and protease at 1% w/w
concentrations.
 Enzymatic pretreatment on
jojoba seeds to facilitate oil
extraction
• A short enzymatic pretreatment of crushed jojoba seeds enabled
centrifugal extraction of more than 25% of the oil contained within
the seed matrix.
• Commercially available hydrolytic enzymes, including glucanases
and proteases, were selected on the basis of their capability to release
soluble sugars and proteins and then examined for their effect on
release of the oil from the seed particles.
• Pectinases and xylanases led to a release of 5-15% of the total sugars
within the seeds, and enabled extraction of 10-15% of the oil.
• Treatment with proteases released 10-15% of the protein contained in
the seeds, but was significantly more effective in releasing 25% of
free oil.
• Only proteases activity caused the entrapment of about 60% of the oil
in the form of extremely stable emulsion.
 Application of enzymatic
pretreatment in bioprocessing

• Bioprocessing Technologies

• Enzymes in bioprocessing

• Potential Area For the Application of Enzymes

in Developing Countries
 Bioprocessing
Technologies
• Fermentation and the use of enzymes, perhaps the
oldest known bioprocessing technologies, and have
been utilised in the preservation of foods for
centuries.
• When applied in food processing, these technologies
are characterised by three major steps: acquisition and
pre-treatment of raw materials, an incubation step to
allow the bioprocess to take place, followed by
product recovery and post-processing.
 Enzymes in bioprocessing
• Enzymes catalyse a number of specific reactions which
produce changes in food constituents, leading to
enhancement of texture, safety, appearance, nutritional
value and flavours in foods.
• Enzymes also serve a diagnostic role as biosensors for
toxicity and quality assessment in food processing.
• Through their antioxidant and biocatalytic activities,
enzymes are applicable as non-thermal food
preservatives.
 Potential Area For the
Application of Enzymes in
Developing
ROLE  EXAMPLE

Upgrading mechanical processing •Fruit juice processing

•Edible oil extraction
•Downstream processing of
starches

Improvement of food safety and •Degradation of phytates

nutritional quality •Degradation of cyanogens
•Degradation of antinutritional
factors
•Enhancement of nutrient density
Non-thermal preservation •Antimicrobial activity
•Antioxidant activity
•"killer enzyme" activity

Acceleration of fermentation •Soy-sauce processing

processes •Fish sauce processing

Diversified use of agricultural •Sugars from starches

products •Brewing with indigenous
grains
•Biofuels from edible oils
 Enzyme Applications For Agro-
Processing in Developing
Countries: An Inventory of Current
and Potential Applications
 Starchy Substrates: Root, Tubers, and Cereal Grains
 Improvement of Nutritional Quality
2. Energy density
3. Nutrient bioavilability
 Detoxification
 Value addition to Roots, Tubers and Cereals Through The
Application of Enzymes
 Sweeteners
 Brewing
 Legumes
 Vegetable oils
 Fruits and vegetables
 Dairy products
 Fish processing
The Use of Enzymes in Fruit
Juice and Cider Processing
 Enzymes used in fruit
•
processing
1. Polygalacturonase (PG): Responsible for the random hydrolysis of 1,4-
alpha-D-galactosiduronic linkages. Depolymerise low esterified pectin (endo -
exo forms).
• 2. Pectin lyase (PL): Cleaves the pectin, by an elimination reaction releasing
oligosaccharides with non-reducing terminal 4-deoxymethyl-alpha-D-galact-4-
enuronosyl residues, without the necessity of pectin methyl esterase action.
• 3. Pectin methyl esterase(PE): Releases methanol from the pectyl methyl
esters, a necessary stage before the polygalacturonase can act fully (the increase
in the methanol content of such treated juice is generally less than the natural
concentrations and poses no health risk).
• 4. Xylanase (hemicellulase): a mixture of hydrolytic enzymes including: xylan
endo-1,3-betya-xylosidase,; xylan 1,4-beta-xylosidase, which degrade
hemicellulose.
• 5. Arabanases (ARA): hydrolyse arabans.
• 6. Ferulic acid esterase (FAE): Cuts ferulic acid and other phenolic linkages
between the xylan chains opening the structure to further degradation by
xylanases.
• 7. Cellulase: Breaks down cellulose.
• 8. Amylases: Breaks down starch.
THANK YOU.