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Encapsulation of Natural Polyphenolic compounds

By

Vaibhav Kumar Maurya

PhD (BAS)

NIFTEM

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Polyphenolic compounds• Secondary metabolites of vascular plants• Derived from the metabolism of shikimic acid and polyacetate

What are the health benefits from polyphenolic compounds?

• Reduce the inflammation by inhibition of the edema• Stop the development of tumors• Present proapoptotic and anti-angiogenic actions• Increase the capillary resistance by acting on the constituents of

blood vessels• Protect the cardiovascular system • Protect retina• Limit weight gain

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Economic implication of polyphenolic compound• Natural coloring agent• Conservative agents • Natural antioxidants • Nutritional additives• Cosmetics and pharmaceutical application

Classes of polyphenol

1. Hydrobenzoic acids

2. Hydroxycinnamic acids

3. Coumaries

4. Stilbenes

5. Flavanols

6. Flavones

7. Flavanones

8. Isoflavones

9. Flavanols

10. Proanthocyanidins

11. Anthocyanins

12. Lignans

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Stability of polyphenol• Instable during processing • Very sensitive to environmental factors

What is encapsulation ?• Shielding to evade chemical reactions or to facilitate the

regulated discharge of core bioactive ingredient from the shell (capsule and coating) with efficient mass transport behavior

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What encapsulation does?• Protect a fragile or unstable compound fro its surrounding

environment• Protect the user from the side effects fo the encapsulated

compound trap a compound (aroma, organic solvent, pesticide essential oil etc)

• Modify the density of a liquid• Change a liquid into a solid • Isolate two incompatible compounds that must coexist in the

same medium • Control the release of the encapsulated compound

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Encapsulation methodPhysical methods Physiochemical

methodsChemical methods

Spray drying Spray coolinginterfacial polycondensation

Fluid bed coating hot melt coating In situ polymerization

Extrusion-spheronization Ionic gelation

interfacial polymerization

centrifugal extrusionsolvent evaporation-extraction Interfacial cross linking

Supercritical liquid method

Simple or complex coacervation

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Spray drying• Very rapid method• Single stage method• Continuous method• Most widely method• Economical, flexible method• High quality and stable particle• Wide particle size range (10-100µm)• Wide selection range for wall material

Wall material• Secondary food grade material, non reactive to

food matrix as well as to bioactive core ingredient used for covering bioactive material

Fig 1: Schematic illustration of a spray- drying apparatus.

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Widely used wall material for polyphenols• Protein (Sodium caseinate and gelatin)• Hydrocolloid (gum arabic)• Hydrolyzed starch ( starch, lactose and maltodextrin)• Chitosan

Natural product encapsulated by Spray drying

• Soybean extract• Grape seed extract• Artichoke• Oak extract• Yerba mate

Fig 2: Scanning electron micrographs of (a) blank microspheres and (b) microspheres loaded with olive tree leaves extract (OLE)

Encapsulation process using supercritical Fluid• Can be classified into

1. As solvent: Rapid Expansion of Supercritical Solution(RESS) and derived process

2. As an anti-solvent: Supercritical Anti Solvent(SAS) and derived processes

3. As a Solute: Particle from Gas Saturated Solution (PGSS) and derived processes

• Supercritical Anti Solvent and Gas Saturated Solution methods are widely used for polyphenol encapsulation

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Supercritical Antisolvent Processing

• Heterogeneous particle size distribution

• Anti solvent dissolves in the solution by decreasing its density and the solvation power of the organic solvent

• Solvent evaporates in the supercritical phase, leading to the oversaturation of the solution, then to precipitation of the solute.

• Excess of solvent is eliminated under a continuous flow of pure supercritical fluid via a purge gate

ss

Fig 3: (A) SEM micrographs of the green tea extract co-precipitated with polycaprolactone (PCL) (MW: 25,000) by Supercritical Antisolvent Process and (B) schematic diagram of the SAS pilot plant

B

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Gas Saturated Solutions Process

• Supercritical fluid plays the role of a solute

• Under pressure gases can be dissolved in liquids

• Gas-saturated solution expanded through a nozzle into the atomization chamber, to form solid particles or liquid droplets

Fig 4: Schematic flowsheet of the particles from gas saturated solutions (PGSS) process

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Application of Gas Saturated Solutions Process• Gentle drying of natural extracts

Fig 5: Scanning electron micrographs of the green tea samples produced by PGSSdrying process

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Physicochemical Methods

Spray coolinghot melt coating Ionic gelationsolvent evaporation-extractionSimple or complex coacervation

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Encapsulation by Cooling of Emulsions• Dissolving or dispersing the active compound in a melted wall material• melted phase is then emulsified in a continuous phase heated at a higher

temperature than the melting point of the coating material• Then environment is suddenly cooled and particles solidify• Allows the microencapsulation of hydrophilic or lipophilic molecules if a

continuous phase is chosen for which these molecules do not have enough affinity

Polyphenolic compound encapsulated by Cooling of emulsion

• Black currant (BCAs) (delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside and cyanidin 3-O-rutinoside

• Epigallocatechin gallate• Quercetin

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Emulsification-Solvent Removal Methods• Evaporation or extraction of the internal phase of an emulsion

giving rise to the precipitation of the polymer coating

Fig 6: Encapsulation by (A) Emulsion/Extraction and (B) Emulsion/Evaporation methods

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Ionic Gelation• Consists of extruding an aqueous solution of polymer through

a syringe needle or a nozzle• Droplets are received in a dispersant phase and are

transformed, after reaction, into spherical gel particles• Chitosan (88.3%)• calcium alginate (90%)• tea polyphenol extract• Elsholtzia splendens extract• Catechin and (-)-epigallocatechin

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Acidic Precipitation• Acid precipitation of the casein• Sodium caseinate • Calcium caseinate• China green tea

Complex Coacervation• Based on the ability of cationic and anionic water-soluble

polymers to interact in water to form a liquid, neutral, polymer-rich phase called coacervate

• Separation of an aqueous polymeric solution into two miscible liquid phases: a dense coacervate phase and a dilute equilibrium phase

• The dense coacervate phase wraps as a uniform layer around dispersed core materials

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Complex Coacervation• Technology parameters are the pH, the ionic strength, the temperature, the

molecular weight and the concentrations of the polymers• Three steps process: formation of an oil-in-water emulsion, formation of

the coating and stabilization of the coating• Particles are not perfectly spherical, and production cost is very high• Useful high value active molecules or for unstable substances• Calcium alginate and chitosan• Pectin and soy protein• Yerba mate extract

Fig 7: SEM microphotographs of control (a) dried in oven and (b) lyophilized beads;and alginate–chitosan (c) dried in oven and

(d) lyophilized beads

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Layer-by-Layer Process• Deposit alternate layers of oppositely charged materials onto mineral

or organic substrates which constitute the core of the particle• Self-assembly technique based on electrostatic attraction of charged

polymers leading to the formation of membranes of controllable thickness according to the number of stacked layers.

1.Polyanion2.wash 1.Polyanion

2.wash

Fig 8. Schematic representation of polyelectrolyte self-assembling

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• Epigallocatechin gallate in polystyrene sulfonate/polyallylamine hydrochloride, polyglutamic acid/poly-L-lysine, dextran sulfate/protamine sulfate, carboxymethyl cellulose/gelatin A

Fig 9. Scheme of gelatin A/EGCG hollow capsule preparation

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Micelles• Based on Hydrophobic Interactions• In an aqueous solution, amphiphilic polymers self-organize

into supramolecular arrangements possessing a hydrophobic central core and a hydrophilic crown

• polymer concentration in solution is higher than the critical micellar concentration (CMC)

• Resveratrol - polycaprolactone (PCL) as the hydrophobic core and poly(ethylene glycol) (PEG) as the hydrophilic shell

• Curcumin- N-isopropylacrylamide (NiPAAm), N-vinyl-2-pyrrolidone and poly(ethylene glycol) acrylate

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Liposomes• Artificial vesicles formed by one

or more concentric lipid bilayers

separated by water compartments• Classes- Small unilamellar vesicles (SUV), large unilamellar vesicles

(LUV) and large multilamellar vesicles (MLV) or multivesicular vesicles (MVV)

• Used to target, protect, release, immobilize or isolate hydrophilic, lipophilic or amphiphilic substances

• Classical Bangham method-hydration of dried phospholipid films• Unstabile in biological fluids and the high speed of active ingredient

release• Payload of the active ingredient is very low• Encapsulation efficiency of phenolic compounds depends on the

morphology of the liposome

Chemical Methods• In Situ Polymerization• Interfacial Polycondensation and

Interfacial Cross-Linking

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In Situ Polymerization• Process consists of emulsifying the monomer component in an

aqueous phase added with an appropriate surfactant• Mostly vinylic and acrylic compounds such as styrene or

methyl methacrylate• Polymerization having been started, the resulting water-

insoluble polymer gives microsphere• Quercetin

Interfacial Polycondensation and Interfacial Cross-Linking

• Chemical reaction by which a membrane made of polymers is created around emulsion droplets

• Reaction occurs at interface between the continuous and dispersed phases. In the emulsion, each phase contains a type of monomer

• Applied to aqueous or organic

active materials

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Fig 10: Principle of the microencapsulation by interfacial polymerization. (A) Theoligomer is soluble in the droplet; (B) the oligomer is insoluble in the droplet

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Formulation parameters• The nature of monomers,• the nature and concentration of the surfactant used• the properties of solvents• the physical parameters of the stirring (speed, time, type of mobile)

Interfacial Cross-Linking• When the water-soluble monomer is replaced by an oligomer or

polymer, this is known as interfacial cross-linking

Fig 11. Mechanism of microcapsule formation by interfacial cross-linking of a hydrosoluble polymer, involving terephthaloyl chloride as an organo-soluble cross-linking agent.

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Interfacial Cross-Linking• Cross-linked grape proanthocyanidin (GPO) • GPO with terephthaloyl chloride (TC) involves hydroxyphenolic groups

leading to the establishment of ester bonds that were detected by infrared spectroscopy

• Cross-linked GPO microcapsules, obtained at pH 9 and 11, had a size lower than 10 μm

Fig 12. Scanning electron micrographs of proanthocyanidin microcapsules (a)

prepared at pH 9.8; (b) prepared at pH 11.

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Other Stabilization Methods• Encapsulation in Yeasts• Co-Crystallisation• Molecular Inclusion• Freeze-Drying

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Encapsulation in Yeasts• Yeast cells (Saccharomyces cerevisiae) as the

encapsulant material• Cells were emptied out of their content by autolysis using a• Clasmolyzing agent (NaCl 5%) and the empty cells were

dispersed in an aqueous phase containing• Chlorogenic acid, and loaded by re-swelling in this solution• Cheap and very effective in terms of loading• Encapsulation of small lipophilic molecules as found in

essential oils• Stable towards a thermal and hydric stress, whereas it did not

hinder the in vitro rele• Chlorogenic acid

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Co-Crystallisation• Introduction the aromatic compound into a saturated solution of

sucrose• Spontaneous crystallization of this syrup is realized at high

temperatures (above 120 °C) and with a low degree of humidity• The crystal structure of sucrose is modified, and small crystal

aggregates (lower than 30 μm) trapping the active molecule are formed

• Granular product have -very low hygroscopicity, a good fluidity and a better stability

• Good economic alternative and remains a flexible technique because of its simplicity.

• Crystals had a size between 2 and 30 μm• Yerba mate extract

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Molecular Inclusion• Appeals to cyclodextrins (CDs). CDs represent a family of cyclic

oligosaccharides consisting of glucopyranose subunits bound through α-(1,4) links

• Three classes: α-, β- and γ-cyclodextrins, composed of 6, 7 or 8 subunits• Cyclic molecules possess a cage-like supramolecular structure, able to

encapsulate various guest molecules• Resveratrol• Live leaf extract• Kaempferol • Quercetin• Myricetin

Figure 14. Molecular model of inclusion complex ferulic acid/α-CD

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Freeze-Drying• Most used processes for the protection of thermosensitive and

unstable molecules• Dehydration operation at low temperature consisting in

eliminating water by sublimation of the frozen product• Polyphenol-rich raspberry

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Thank you