טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentIsaschar Ovdat Sivan
SubjectCrosslinking Soy Proteins with Tyrosinase from Bacillus
Megaterium for Modulating Functionality
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Ayelet Fishman
Full Thesis textFull thesis text - English Version


Abstract

The overall goals of this research were (a) to investigate the effect of tyrosinase-mediated crosslinking of soy protein-based emulsions and gels, and (b) study the underlying mechanism behind the enzyme’s crosslinking activity using glycinin-derived peptides. Tyrosinase from Bacillus megaterium (TyrBm) was previously isolated and characterized in our lab. This research utilizes TyrBm as a biological crosslinking tool for the modulation of soy proteins for potential food applications.

TyrBm was used to crosslink the two main soy proteins, glycinin and β-conglycinin (BCG), as well as commercial soy protein isolate (SPI). Optimization of the reaction conditions enabled to achieve for the first time crosslinking without a mediator, directly through the protein tyrosine residues. Crosslinked protein was used to fabricate oil-in-water emulsions that were studied for their physical stability, particle size and viscosity. Each protein fraction showed different properties following crosslinking. While the TyrBm-crosslinked glycinin emulsion exhibited a two-fold reduction in creaming velocity, a similar increase in viscosity and an overall improved emulsion physical stability, the two other TyrBm-treated emulsions (BCG and SPI) showed deteriorated stability after the enzymatic reaction. It was determined that the enzymatic crosslinking is an important and effective tool when used on poor emulsifiers as it leads to increased particle size thus reducing mobility and phase separation. The crosslinked glycinin formed a more stable emulsion with a gel-like structure while the BCG and SPI crosslinked emulsions showed crosslinked particles however their size did not lead to a gel-like structure but rather to induced flocculation and phase separation.

Heat-induced soy glycinin gels were evaluated for their rheological behavior and texture properties. TyrBm-crosslinked glycinin gels possessed a 330-fold higher storage modulus (G') and a 2-fold increase in hardness and gumminess compared to the non-crosslinked control. SEM imaging linked these macroscopic phenomena to a 3-fold increase in pore diameter. In addition, a reduced sugar and fat soy-based chocolate pudding was fabricated. The crosslinked soy-based pudding exhibited higher hardness than the non-crosslinked pudding. A tasting panel reported a significant positive correlation between the crosslinked soy pudding and measures of "stable" and "firm" texture. These results confirm that TyrBm-crosslinking of soy proteins can affect the product texture in a complex food matrix.

The crosslinking mechanism of TyrBm was studied by investigating three tyrosine-containing octapeptides derived from the glycinin protein sequence. The effect of the location of the tyrosine residue in the amino acid sequence and of its surrounding amino acids was evaluated. A significantly lower activity was measured when tyrosine was located in the middle of the peptide chain than at the C- or N-terminus. Mass spectra analysis revealed that crosslinking is achieved via the oxidative quinone ring of the tyrosine residue by aryl-alkylamine addition or aryloxy radical coupling and a numerical correlation was found between the monomer and oligomer molecular weight. This work confirms that protein crosslinking occurs by several chemical mechanisms leading to different products.

Overall, the study demonstrates the rational development and application of bacterial tyrosinase as a technological aid for the improvement of food formulations' stability and texture.