טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMoscovici Alice Maayan
SubjectImpact of Maillard Reaction on the Digestibility and
Prebiotic effects of Milk Protein-Carbohydrate
Mixtures
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Uri Lesmes
Full Thesis textFull thesis text - English Version


Abstract

Food processing offers great potential to exhaust the beneficial effects of food ingredients and minimize deleterious ramifications to health and well-being. Thermal processing of protein-carbohydrate mixtures may result in Maillard reaction, consequently altering protein functionality. Nevertheless, gaps exist in understanding how this reaction can be rationally harnessed to modulate digestibility, particularly the generation of bioactive peptides and gut microbiota. Thus, this research aimed to holistically study the digestive fate of Maillard reaction products (MRPs) throughout the digestive tract of an adult or infant.

This work focused on bovine lactoferrin, α-lactalbumin and β-lactoglobulin, viable rich sources for bioactive peptides, e.g. antimicrobial, immune-modulatory and bifidogenic, and on prebiotic fructo-oligosacchrides (FOS) and galacto-oligosaccharides (GOS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and absorbance (305 nm) indicated the formation of MRPs, further supported by color development, an important Maillard reaction characteristic. In addition, mean colloidal size and turbidity determined at 3<pH<10 revealed decreased colloidal stability compared to the native proteins, particularly around the isoelectric point, which was not altered following conjugation. Moreover, lactoferrin-FOS conjugation was more extensive in solution compared with powder form, and GOS (aldose) was more reactive than FOS (ketose) with α-lactalbumin under ?dry‟ conditions.

Next, native proteins, controls and MRPs were subjected to in vitro gastro-duodenal models recreating the digestive conditions of a healthy adult or infant. Proteolysis was monitored by SDS-PAGE, which demonstrated proteins and MRPs were more susceptible to adult conditions compared to infants. Interestingly, infant digestion revealed a mixed trend, as FOS-based MRPs initially resulted in a decrease in both lactoferrin's and α-lactalbumin's proteolysis extent, while prolonged heating led to enhanced susceptibility. Proteomics of lactoferrin infant digesta pointed-out altered enzymatic cleavage patterns. Data mining revealed samples contained fragments derived from bioactive peptides, e.g. antimicrobial lactoferricin and lactoferrampin, with homology up to 89% as well as 100% homology with several peptides reported in vivo. Furthermore, bioinformatics enabled the identification of 36 potentially novel bioactive peptides formed during MRPs digestion with bioactivity probability of up to p=0.89, two of which (WIIPMGILRPYL and FGSPPGQRDLL with p=0.77 and 0.80, respectively) were also identified in vivo.

Finally, the fermentability of native lactoferrin, heated or conjugated with FOS was studied. Samples were predigested in an in vitro gastro-intestinal adult model and fed to an ex-vivo batch colon model inoculated with human feces. Metagenomic analyses demonstrated the various samples altered gut microbiota, but produced a similar bacterial profile. Moreover, Bifidobacteriaceae/Bifidobacterium relative abundance was lower in predigested MRPs vessel compared to FOS, suggesting that conjugation diminished its prebiotic effect.

In conclusion, data obtained in this study highlights the Maillard reaction could have a mixed impact on proteolysis extent, resolving the contradicting evidence in literature, and also potentially modulate the generation of bioactive peptides during digestion. Furthermore, this study presents a new paradigm for analyzing Maillard reaction impact on digestibility, monitoring the peptide sequences liberated and their bioactivity as well as comparing digestibility in different age-groups. Altogether, this research provides a holistic approach to facilitate the reverse engineering of protein-carbohydrate products rationally designed to benefit the consumer.