טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShani Levi Carmit
SubjectDevelopment of Dynamic In Vitro Gastrointestinal Models
and Their Application to Study Protein and
Hydrocolloid Digestion
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Uri Lesmes
Full Thesis textFull thesis text - English Version


Abstract


The mass production of industrial or processed food has supported the development of human society accommodating the modern lifestyle we now know. Such massively produced products offer various benefits to the consumer, e.g. delivery of health benefits even beyond a food's nutritional value. The concept of reverse engineering of such foods based on their gastrointestinal behavior has led to an upsurge in studies looking into the physicochemical basis of digestion and application of various in vitro methods as robust tools of research.

The overall goal of this research was to develop and apply in vitro GIT models to study the physicochemical basis of novel milk-protein based food delivery systems in different human populations. The first model was designed based on auto-titration unit to be a semi-dynamic system mimicking gastric pH gradients found in the adult and infant stomach. This model was used to examine the proteolysis of β-lactoglobulin and lactoferrin and the behavior of their corresponding emulsions as well as Pickering-type emulsions (stabilized by lactoferrin based nano-particles and dietary fibers). SDS-PAGE of digesta collected from the semi-dynamic model revealed significant differences in protein breakdown compared to the common in vitro gastric model held at a constant pH. Interestingly, b-lactoglobulin was found to breakdown faster under infant compared to adult conditions, contrary to lactoferrin.  Regarding emulsion behavior, laser based droplet sizing and fluorescence microscopy provided new insights into emulsion destabilization during gastric digestion and revealed differences in rate and mechanisms of instability (droplet coalescence versus floculation) between adults and infants. In addition, Pickering emulsions stabilized by lactoferrin nano-particles were studied to and revealed nano-particles may increase emulsion stabiulity whithout significanlly altering droplet sizes and modulate emulsion responsiveness to the conditions of the mouth and stomach.

The second model was developed to be a highly bio-relevant dynamic in vitro model based on two serially connected bioreactors recreating various dynamic aspects of the adult or elderly alimentary canal. Uniquely, a comprehensive literature review enabled identification of physiological parameters, such as the pulastile nature of gastric mixing, the rate of gatric emptying and the dynamics of bile secretion. These were applied in the set-up of the system, the physicochemical parameters applied and the development of the controlling software. SDS-PAGE and LCMS analyses of samples collected from in vitro digestion of β-lactoglobulin, α-lactalbumin and lactoferrin suggest the bioaccessibility of "slow-digesting" and "fast-digesting" proteins identified in adults do not necessarily maintain this trait under elderly gastro-intestinal conditions. This model was also used to demonsrate gastric lipase is essential in emulsion breakdown and a factor distinguishing emulsion breakdown in the gut of adults and the elderly.

Overall, this study highlights the importance of recreating the dynamics of human digestion when assessing oral formualtions in vitro. Moreover, the study brings forward two innovative generic yet advanced models recreating various aspects of human digestion. These could help shed light into the underlying principles governing the digestive fate of loral liquid-formulations and facilitate age-tailoring their digestive fate.