טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentCohen Yifat
SubjectBioaccessibility and Bioavailability of Hydrophobic
Nutraceuticals Encapsulated within Protein
Nanoparticles
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yoav Livney


Abstract

Lipophilic nutraceuticals are typically found and consumed within a fat phase, which our gastrointestinal system has evolved to suitably digest. Using novel protein-based nano-vehicles for lipophilic nutraceuticals raises important questions regarding the bioavailability of the entrapped nutraceutical, and its absorption mechanism in the absence of fat. To address these questions, we focused on vitamin D3 (VD3) loaded re-assembled casein micelles (rCM) as a model system. In-vitro gastro-intestinal digestion of the VD3 loaded rCM was performed to study rCM digestibility and VD3 retention. HPLC analysis of digesta revealed that VD3 retention is significantly higher when it is encapsulated in rCM compared to free VD3 (85% and 15% after 3h respectively). These high retention rates combined with in-vitro bioavailability, examined using Caco-2 cells, indicated an improved oral bioavailability of the encapsulated VD3 compared to the unencapsulated VD3.

RCM were found to protect VD3 during simulated adult and elderly digestion conditions (94% and 85% VD3 retention, respectively). 

To examine the effect of nano-encapsulation within rCM, and of the absence of fat on the bioaccessibility and bioavailability of VD3, and its absorption mechanisms, non-fat yoghurt was chosen as a model food system. Three yoghurt formulations were prepared: 3% fat yoghurt enriched with VD3 dissolved in milk-fat (“VD3 in the fat”), non-fat yoghurt and 3% yoghurt enriched with VD3 encapsulated within rCM (“VD3 in the protein, in the absence and presence of fat, respectively”). The yoghurts underwent in-vitro oral-gastro-intestinal digestion. VD3 retention and bioaccessibility were high (~90% and ~70% respectively) in all formulations. VD3 uptake by Caco-2 cells from the digesta was three-fold higher for the non-fat yoghurt enriched with VD3 in rCM compared with the 3% fat yoghurts enriched with VD3. Zeta-potential of the bioaccessible fraction of the digesta was more negative in the presence of fat, as fatty acids in mixed micelles increased their negative charge, thereby increasing their repulsion from the negatively-charged intestinal cell membrane, causing lower VD3 uptake from the fat containing samples. SR-BI, CD36 and NPC1L1 transporters were found to be involved in VD3 absorption from the digesta obtained from the three yoghurt formulations, as examined in vitro using cell cultures. We concluded that when VD3 is in rCM, in the presence of fat, VD3 competes with lipolysis products, as ligands for these transporters. Hence, VD3 uptake from rCM in the presence of fat was lower.    

A large scale (94 healthy volunteers) double-blind placebo-controlled clinical trial was performed to examine how the bioavailability of VD3 is affected by its delivery in protein nanoparticles, rather than in the fat, and in its presence/absence. Yoghurts of the three formulations, in addition to a placebo formulation of unenriched non-fat yoghurt, were prepared. The results of this single high dose ingestion clinical trial indicated that the bioavailability of VD3 in protein based nanoparticles in the absence of fat did not significantly differ (p>0.1) from its bioavailability in fat.

This work provides pioneering evidence that protein-based nano-vehicles present an excellent alternative to fat for delivery of hydrophobic nutraceuticals in foods.