טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentIsraeli Lev Gal
SubjectControlling Crystallization of Hydrophobic Bioactives
Using Proteins:Towards Application in Delivery
Systems
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yoav Livney
Full Thesis textFull thesis text - English Version


Abstract

Certain proteins possess the intriguing ability to affect nucleation and growth of crystals. Proteins adsorb onto solid surfaces through various interactions, predominantly, hydrophobic, van der Waals and electrostatic interactions. Selective adsorption to certain crystal faces modifies crystal morphology, while non-specific protein adsorption to all of the crystal planes, inhibits crystal growth dramatically, and may be useful for controlling crystal size while nano-entrapping it.

Protein-surface and protein-crystal interactions are becoming increasingly important in many areas including drug and nutraceutical delivery, as many nutraceuticals and pharmaceuticals are highly hydrophobic and crystalline in nature, resulting in low water-solubility and poor bioavailability. The improved ability to control lipophilic bioactive nanocrystal formation and dissolution can increase stability and loading capacity, and open new ways to control the release of incorporated bioactives and their bioavailability.

The main goal of this project was to study the relationship between protein structure and its efficacy in controlling crystal size, morphology and in-vitro bioavailability, by comparing three model proteins: β-casein, hydrophobin, and β-lactoglobulin, representing different structural groups of proteins. We assessed their functionality in preventing crystal growth, using genistein as a model hydrophobic crystallizing bioactive. We also assessed the proteins interaction with other model hydrophobic bioactives: vitamin D3 and naringenin. We formed nanoparticles consisting a hydrophobic bioactive stabilized by a protein; characterized the nanoparticles size, morphology, crystallinity and the interaction between the protein and the crystal; evaluated the protection conferred to the bioactive against oxidation; and quantified the effect of each of the three proteins on the in-vitro bioavailability of genistein.

β-casein and β-lactoglobulin were previously studied in our lab for their function as nanoencapsulation agents for food enrichment and drug delivery. Hydrophobins' application in nanoencapsulation of nutraceuticals for food enrichment has apparently not yet been explored. We showed that hydrophobin tends to self-assemble in aqueous solution, and bind hydrophobic substances. Vitamin D3-hydrophobin complexes are nanoscale, their aqueous solution is transparent, thus they may be suitable for enrichment of clear beverages. Additionally, Hyd provides excellent protection to Vitamin D3 against degradation.

Dynamic light scattering, polarized light microscopy and cryo-TEM showed that β-lactoglobulin, hydrophobin and β-casein, inhibit genistein crystal growth in aqueous solution in increasing order of efficacy. Protein structure determines the mechanism and the efficacy by which it affects crystal growth and morphology: β-lactoglobulin, a rigid globular protein with an inward facing hydrophobic domain, indirectly suppresses crystallization by binding and reducing concentration of free hydrophobic compound molecules. Hydrophobin, a rigid globular protein with a flat external hydrophobic domain, adheres to the surface of certain crystal faces limiting growth in the perpendicular directions, thereby modifying crystal morphology. β-casein, a rheomorphic protein with an external hydrophobic domain, adheres to different crystal faces nonspecifically, thereby blocking growth in all directions, resulting in round coated nanocrystal formation. Consequently, an inverse correlation was observed between nanocrystal size and in-vitro bioavailability. Based on this study, amphiphilic proteins can be more effectively selected and applied to control crystal growth and morphology of hydrophobic bioactives to improve their delivery and bioavailability in food and drug systems.