טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentNadav Ron
SubjectBeta Lactoglobulin as a Nano-Capsular Vehicle for
Hydrophobic Nutraceuticals
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Livney Yoav
Full Thesis textFull thesis text - English Version


Abstract

    β-lactoglobulin is the major whey protein of ruminant species. β-Lg is a globular protein of 18,400 Da Mw and 162 amino acid residues. The structure of β-Lg contain 8-stranded, antiparallel β-barrel with a 3-turn α-helix on the outer surface and a ninth β-strand flanking the first strand. The so called calyx, or β-barrel, which is conical, forms the main ligand binding site, and evidence point out that there is a second ligand binding site between the α-helix and the β-barrel of the protein surface at the external pocket.

    The goal of this research was to evaluate the capability of β-Lg to bind hydrophobic nutraceutical substances which have not yet been reported as its ligands (lycopnene, CLA, DHA), and to develop a process for forming nanoparticles comprising the ligand-containing β-Lg and a secondary protection by polysaccharides which are electrostatically complexed with the protein.

    Spectrofluorometry method couldn’t detect Lycopene, CLA and DHA binding to β-Lg, however, using ITC we showed that β-Lg can bind CLA and DHA with binding constants (K) of 2.3*105 and 6.3*104 respectively. Those findings indicated that the binding site does not include the trp, therefore it is likely that the binding site is outside the calyx.

        By varying chitosan protein ratio, we managed to form nano‑complexes which were stable, but not small enough to form a transparent solution.

    By studying complexation of β-Lg and pectin, we found a ratio range which gave the desired particle size (less than 150nm) and the solution was transparent enough.

    We studied the protective effect of the complex on vitamin D2 and found that the vitamin concentration inside the complexes was 55 time higher than it’s concentration in the serum, and 5.7 times higher than the initial concentration dissolved in the solution. We have demonstrated a better protective effect to the ligand when pectin was used for the nanoencapsulation, compared to encapsulation in  pure b-Lg (58% after 120hr Vs. 37% using b-Lg alone) .

    In conclusion, using ITC we have shown for the first time that β-LG can bind CLA and DHA. We have developed a novel “nanocoating” technology based on forming electrostatically stable nanoparticles, of about 100 nm in size, using β-LG and an anionic polysaccharides (pectin). Complexes can be made with a cationic polysaccharide as well, although further study would be required to achieve small enough particles for transparent drinks.