Encapsulation of Food Antioxidants as Potential Functional Food Ingredients Amyl Ghanem Ph.D. P.Eng.

Chemical Engineering Dalhousie University

Health benefits of plant polyphenols

– Plant polyphenols possess a high spectrum of antioxidant, anti inflammatory anti bacterial and antiviral functions.

– Research suggests that plant polyphenols can slow the progression of certain cancers, reduce risk of cardiovascular disease, neurodegenerative disease, diabetes, osteoporosis etc.

Challenges

• • • Concentrations that are effective in vitro are often an order of magnitude higher than in vivo.

Low bioavailability of polyphenols • small proportion of the molecules ingested orally actually make it into bloodstream • short gastric residence time, low solubility/permeability in the gut or degradation due to enzymes, pH etc. in the GI tract.

Instability of molecules during food processing and storage • degradation due to exposure to light, oxygen, temperature.

“Microencapsulation*”

Technically the formation of a wall material around a core/active material to make a capsule, on the scale of 1-1000 microns. However this term has come to encompass “entrapment” as well, Which includes the distribution of the core/active material within a matrix: wall matrix Core/active active • • • • • Objective: Protect the core/active material from degradation in storage, processing or active conditions Improve bioavailability, cell uptake of core/active material Act as a slow release reservoir Improve solubility of core/active material target delivery of core/active material to a specific location *Nanoencapsulation applies similarly to a nano-size range

• • • • •

Encapsulate or entrap plant extracts in microparticles/nanoparticles

Achieve high concentration of active molecules in small volume.

Matrix material would stabilize polyphenols during storage and processing.

Matrix material could be used to improve bioavailability.

Applications exist in food, pharmaceutical and cosmetics industries.

Aim for particle size range < 30 m m or even lower not affect texture or clarity.

My background:

Entrapment of molecules for Drug Delivery Systems Purpose: to understand and manipulate the fate • • of drugs in humans.

Controlled release Tissue targeting Aicello Japan Designed to release drug in the small intestine

Encapsulation Methods

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Spray drying

Widely used in the Food Industry Common wall materials: • Modified starch • Maltodextrin • Gum Arabic Spherical particles 10-100 m m Limitations: wall materials, high T

Freeze-Drying

Dehydration process good for heat sensitive materials Active material and matrix material in solution Results in powder of “uncertain form” Great potential to combine with other methods

Cloudberry extract with Maltodextin Lane et al, Agricultural and Food Chemistry 2008:11251-11261

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Ionic interactions: Coacervation, Gelation

Active molecule + matrix material Counter ion solution Microcapsules with entrapped active molecule Gel in solution deposits around the • • • active ingredient which is suspended Gelatin Calcium alginate Chitosan Considered expensive but does not involve high temperatures or solvents.

Control sizes from nano to micron sized

-Liposomes

Lipid bilayer membrane encapsulating an aqueous phase Formed from phospholipids utilizing hydrophobic/hydrophilic interactions Formed by: thin film evaporation, sonication, reverse phase evaporation, melting, freeze thawing, extrusion A lot of literature on this technique Shown to improve bioavailability and targeting Often low entrapment efficiency and loading Rapid release of active material Can be improved by coatings

Fang and Bandhari, Trends in Food Science and Technology 21(2010) 510-523

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Inclusion Complex

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Using cyclodextrin as an encapsulating material

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Hydrophobic/hydrophilic areas helps to improve the water solubility of molecules.

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Emulsification

• • • •

Active material dispersed into matrix/wall material emulsified and cooled; Or evaporation of internal phase Lipids, hydrophilic polymers such as gelatin, glucan or agarose

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Thermal gelation

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Supercritical fluid

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Combinations of techniques, crosslinking, coatings etc.

Fang and Bandhari, Trends in Food Science and Technology 21(2010) 510-523

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My background: Entrapment of molecules for Drug Delivery Systems

Matrix material: chitosan • Active material – BSA (sample protein) – Glucose oxidase (sample enzyme) – Cladribine, adenosine (nucleotides, anticancer drug) – bFGF (growth factor) • Methods: – Complex coacervation of CH and TPP – Crosslinking with gluteraldehyde, glyoxal, genipin)

Chitosan Nanoparticles (CH NP)

Unmodified, unloaded CH Nanoparticles (112 nm  13) Unmodified chitosan loaded with 100 ng of bFGF 87,000 x magnification (157 nm  23) N-succinyl Chitosan, unloaded, dried Magnification 16,500 × (642 nm  90)

Particle Properties

• Sizes: Microparticles and Nanoparticles – 100-150 nm when dried (swell to 500 nm) – Smooth spherical morphology – Some aggregation observed • Good loading efficiencies – 70% for cladribine (anticancer drug) – 50% for bFGF (growth factor) • Can manipulate to modify behaviour – Crosslinking (ionic, glyoxal, genipin) – Modification (N-succinyl chitosan)

Controlled Release

Overall release from crosslinked Cladribine-loaded nanoparticles into PBS, pH 7.4.

No entrapment 180 160 140 120 100 80 60 40 20 0 0 Genipin Crosslinked (2 h, 0.1 mg/mL genipin) Glyoxal Crosslinked (2 h, 50 mg/mL glyoxal) 20 40 60

Time (h)

80 100 120 Domaratzki, A and Ghanem, A. Journal Applied Polymer Science 2013, 128: 2173–2179 18

25.0% 20.0% 15.0% 10.0% 5.0% 0.0% 0 bFGF release from nanoparticles into PBS, pH 7.4

chitosan CH CH+heparin SCH SCH+heparin N succinyl chitosan +heparin 50 100

Time (hrs)

150 200 250 19

Examples of Polyphenol Entrapment

Encapsulation of anthocyanin extract from jabuticaba and storage stability Extracts by supercritical CO 2 , compared storage conditions of 3 systems at 14 days :

Encapsulation Efficiency Degradation 25 oC light Degradation 25 oC No light

60% ~50% Free anthocyanin Polyethylene glycol Ca-alginate system 79.78% 98.67% 60 25 30 20

Degradation 4o No light

~50% ~0 ~0 Both encapsulated systems were more stable under light and temperature Santos et al, Food Research International, 2013:617-624

• • •

Spray drying of blueberry extract

Freeze dried blueberry, and blueberry pomace extracted into acetone (A), ethanol (E) or methanol (M) Spray dried with whey protein or gum arabic Subjected to in vitro digestion model

Encapsulation Efficiency (TPC) Antioxidant activity during digestion (Frap % 2hours) Antioxidant activity during digestion (Frap %4hours )

Gum Arabic A E M 68 106 95 ~65 87 90 ~30% 35 30 Whey Protein A E M 53 137 102 65 85 85 55 80 85 No comparison to Free extract However they did show that WPI preserved antioxidant activity during simulated digestion Flores et al, Food Chemistry, 2014:272-278

Possible applications to Haskap

• • • • • Currently investigating extraction of blueberry polyphenols and encapsulation by spray drying and freeze drying Steps: what concentration can be achieved in the extract? Recommend a combination of methods to achieve high entrapment, stability and bioavailability Main variables would include material(s), extraction method for polyphenols, encapsulation Encapsulation facilities – spray drying, freeze drying, liposome formation, coacervation

References

• • Encapsulation of Natural Polyphenolic Compounds: A Review. Munin, A. and Edwards-Levy, F. Pharmaceutics. 2011:793-829.

Encapsulation of Polyphenol- a Review. Fang, Z and Bhandari, B. Trends in Food Science and Technology 2010:510-523.