Derek Dee

Food protein nanofibrils may have improved functionalities in food applications owing to their highly ordered structures and extreme aspect ratios. Protein fibrillization is typically inducible at high temperature and low pH, where proteins are hydrolyzed and self-assemble into fibrils spontaneously. This process may be accelerated by seeding with fragments of pre-formed fibrils. As plant proteins are becoming more prevalent as environmentally sustainable and healthy alternatives to animal proteins, more research has begun to focus on legume protein fibrillization. Lentils are a major Canadian pulse crop with high protein content, but little is known about lentil protein fibrillization. The objectives of this project were to evaluate (1) the effects of protein extraction conditions (acidic vs. alkaline pH), (2) seeding with pre-formed fibrils on lentil protein fibrillization, and (3) the gelation of lentil protein fibrils. Lentil proteins extracted at either pH 8.2 or pH 3.5 with salt were incubated at 80 °C, pH 2, with stirring, to form fibrils. The protein composition and hydrolysis pattern during fibrillization and the fibrillization kinetics of the two protein extracts were found to be similar. However, transmission electron microscopy revealed apparent morphological differences—fibrils made from alkaline extract were more heterogeneous, curly, and tangled, whereas fibrils made from acidic extract were more uniform, long, and straight. Further analyses with UV-vis spectroscopy, the Fast Blue BB assay, and LC-MS indicated the presence of protein-phenolic interactions in the alkaline extract, which likely affected fibrillization. The acidic extracts were used for further studies of fibrillization. The lag time, growth rate, conversion yield, and fibril morphology were evaluated for seeded and unseeded fibrillization. Seeding with 3% w/w seeds significantly decreased the lag time (p
View record

Legume seed storage proteins can be induced to form amyloid fibrils by heating at low pH, which could improve protein functionality for use in food and materials applications. However, amyloidogenic regions of legume proteins are largely unknown. In this study, crude protein extracts and the major seed storage proteins, 7S and 11S globulins from pea and soy were enriched and induced to form fibrils at pH 2, 80°C. The hydrolysis, fibrillation kinetics and fibril morphology were characterized by gel electrophoresis, Thioflavin-T (ThT) fluorescence assay and transmission electron microscopy (TEM). The amyloidogenic core regions of pea and soy 7S and 11S globulins were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). It was found that all samples were further hydrolyzed and fragmented into shorter fibrils during extended heating (30 – 78 h). 7S globulins from either pea or soy displayed no lag phase whereas 11S globulins and crude extracts exhibited a similar lag time of around 8 h. Pea and soy protein fibrils demonstrated morphological differences where most pea protein fibrils were straight and soy protein fibrils were rather worm-like. Over 100 unique peptides were detected from the fibril core of pea 7S globulin, and over 50 unique peptides found in pea 11S globulin, soy 7S and 11S globulin fibril core, most of which could be mapped on several conserved fibril core regions. Amyloidogenic regions were restricted mainly to the homologous core region of 7S globulins and the basic subunit of 11S globulins. Overall, pea and soy 7S and 11S globulins are rich in amyloidogenic regions. This study further informs our understanding of legume protein fibrillation mechanism and engineering food protein fibrils with specific structures and functionality.

View record

Amyloid fibrils are gaining attention as novel food ingredients and nanomaterials, due to their unique structural and chemical properties, and functionality as stabilizers and gelling agents. Amyloid fibrils are long, thin, unbranched protein aggregates that are important in medicine, biology and nanotechnology. Many proteins, including food proteins (e.g., from milk, eggs, and legumes), can be converted into amyloid fibrils under the proper conditions, most commonly by heating at low pH. Most of the research on nanofibrils has dealt with animal proteins, so a fundamental understanding of the self-assembly, structure, and functionality of nanofibrils derived from plant proteins is lacking. Plant protein is considered a sustainable source of protein. However, the functional properties of plant proteins, such as foaming, gelling, and emulsification, are generally inferior to that of animal proteins. Consequently, one method to improve the functionality of plant proteins is to produce nanofibrils, which are promising materials.The goal of this research was to relate nanofibril structure and functionality by comparing nanofibrils made from various legume proteins. Nanofibrils were formed from peanut, lentil, pea, and mung bean during incubation at pH 2 and 85 °C. The results showed that protein extracts from peanut, mung bean, pea, and lentil formed nanofibrils, which were detected using thioflavin T and transmission electron microscopy (TEM). SDS-PAGE revealed that extensive protein hydrolysis occurred during the onset of fibril formation, indicating the significance of hydrolysis to fibrillation under these conditions. Using TEM, fibrils from different legumes showed morphological variability with differences in length, width, and flexibility. This research revealed that peanut, lentil, and mung bean fibrils were most soluble at pH 2 and least soluble at isoelectric point (pI) pH. Also, the fibrils showed smaller particle size at pH 2 to that of pH 7 which is consistent with the solubility result. The presence of the fibrils results in an increase in viscosity compared to the unheated samples. The findings showed that a better understanding of legume fibrils is needed to increase their usage as functional materials in food systems, and that this would probably extend theoretical knowledge of the structure-function relationship between plant-based fibrils.

View record