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 PercolationThis 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 Traditional Extraction

• High temperature during water extraction destroys the important heat labile active ingredients present in the herbal extract.

• All the bioactive ingredients present within the cell walls are not able to get extracted to their optimum level due to hard cement bounding present within the cell wall.

• The color, flavor, aroma, and consistency of the final herbal extract are never consistent from batch to batch.

Advantages of enzymatic treatment over other methods of extraction

• Enzymatic treatment for extraction is different from solvent extraction since enzymes are biospecific and attack only on their sustrates.

• Due to the specificity of enzymes, there is less chance of degradation of the phytoconstituent and also small quantities of enzyme can act on large quantities of substrate.

• Enzymatic action damages cell wall, favouring permeability of phytoconstituent especially the constituent of interest.

• The profitable effects of utilizing enzymes have long been recognized, with regard in the increasing yields of the main products by reducing side products and low waste treatment.

• The extraction using solvents are generally carried out at high temperatures,which may be little or of no use if the compound of interest is not thermostable.therefore,

• Another advantage is the tailoring of enzyming complexes to fit the processing requirements because of the mild conditions that avoid drastic operational conditions.also use of enzyme is circumvented; as 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:

1. Enzyme preparations may consist of whole cells, parts of cells, or cell-free extracts from the source used.

2. Active components have known molecular weights that range from 12,000 to several hundred thousand.

3. Enzymes may be in liquid, semi-liquid, or dry form. An enzyme in general and pectinase in particular is readily soluble in water.

4. 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).

5. Dry preparations are off-white to tan amorphous, finely divided powders.

6. 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 Benefits

CelluSEB-TL Cellulase used for eetraction, for liquifaction of plant materials and for downstream processing

· 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

SEBstar-HTL Heat-stable alpha-amylase used for high temp. liquefaction of starch containing grain mashes

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

grain starch to glucose

· Excellent thermal and pH stability· 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

SEB-Neutral PL

Protease used for conversion of grain proteins to increase free-amino nitrogen

· Hydrolyzes both animal and vegetable proteins· Breaks down and increase protein dispersibility, solubility,

palatabilitiy and digestability

SEBferm-L A blend of amylases and proteases used for the simultaneous saccharification and fermentation of grain starch.

· Improved yeast growth and faster fermentation rate· Higher alcohol yields and DDG protein value· Reduced thin stillage viscosity 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 Bioprocessing Technologies 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 nutritional quality

•Degradation of phytates

•Degradation of cyanogens

•Degradation of antinutritional factors

•Enhancement of nutrient density

Non-thermal preservation   •Antimicrobial activity

•Antioxidant activity

•"killer enzyme" activity

Acceleration of fermentation processes

•Soy-sauce processing

•Fish sauce processing

Diversified use of agricultural products

•Sugars from starches

•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 Quality1. Energy density2. 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.

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