1. Encapsulation of Food Antioxidants as Potential Functional Food Ingredients Amyl Ghanem Ph.D. P.Eng. Chemical Engineering Dalhousie University

2. 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.

3. 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.

4. 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 “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 Core/active matrix active *Nanoencapsulation applies similarly to a nano-size range

5. 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 or even lower not affect texture or clarity.

6. My background: Entrapment of molecules for Drug Delivery Systems Aicello Japan Purpose: to understand and manipulate the fate of drugs in humans. Controlled release Tissue targeting Designed to release drug in the small intestine

7. Encapsulation Methods – Spray drying Widely used in the Food Industry Common wall materials: Modified starch Maltodextrin Gum Arabic Spherical particles 10-100  m Limitations: wall materials, high T

8. Lane et al, Agricultural and Food Chemistry 2008:11251-11261 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

9. – 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

10. -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

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

12. – Inclusion Complex Using cyclodextrin as an encapsulating material Hydrophobic/hydrophilic areas helps to improve the water solubility of molecules. – 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 – Thermal gelation – Supercritical fluid – Combinations of techniques, crosslinking, coatings etc.

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

14. 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) My background: Entrapment of molecules for Drug Delivery Systems

15. Chitosan Nanoparticles (CH NP) 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) Unmodified, unloaded CH Nanoparticles (112 nm  13)

16. 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)

18. Overall release from crosslinked Cladribine-loaded nanoparticles into PBS, pH 7.4. Domaratzki, A and Ghanem, A. Journal Applied Polymer Science 2013, 128: 2173–2179 No entrapment 18 Controlled Release

19. bFGF release from nanoparticles into PBS, pH 7.4 chitosan +heparin N succinyl chitosan 19

20. Examples of Polyphenol Entrapment

21. 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 Degradation 4o No light Free anthocyanin -60%~50% Polyethylene glycol 79.78%6030~0 Ca-alginate system 98.67%2520~0 Both encapsulated systems were more stable under light and temperature Santos et al, Food Research International, 2013:617-624

22. 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 55 80 85 Flores et al, Food Chemistry, 2014:272-278 No comparison to Free extract However they did show that WPI preserved antioxidant activity during simulated digestion

23. 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 Possible applications to Haskap

24. 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.