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Extraction of canola proteins and assessment of their applications: cruciferin for encapsulation of bioactives and napin as a chaperone-like molecule

  • Author / Creator
    Akbari, Ali
  • Canola is a farm-gate crop in Canada. Canola meal after oil extraction is used mostly as animal feed with limited value-added applications. Canola proteins are known to have great potential for use in food and non-food applications due to their nutritional, biological and functional properties. Canola contains two major proteins: cruciferin and napin, but with distinct properties. Cruciferin is resistant to gastric digestion and has excellent functional properties; it was hypothesized that it might be an appropriate material for encapsulation of bioactive compounds. Napin, a small molecule that is extremely thermal resistant, may represent chaperon-like activity. Therefore, the overall objectives of the thesis are 1) to develop an integrated method of extraction for these two main canola proteins, 2) to explore the potential application of cruciferin for encapsulation of bioactive compounds, 3) to study in vitro cellular uptake and trans-cellular transport of cruciferin-based particles, 4) to evaluate the potential chaperone-like activity of napin. An integrated, simple and scalable method, including acidic washing (pH 4), alkaline extraction (pH 12.5), isoelectric precipitation (pH 4), and ultrafiltration, was developed to isolate two main canola proteins: cruciferin and napin. Negatively-charged Cruciferin/calcium (Cru/Ca) nanoparticles were prepared using a cold gelation method. To study the potential application of cruciferin to coat and protect chitosan particles at stomach low pH, positively-charged spherical cruciferin/chitosan (Cru/Cs) nanoparticles were also prepared. Cru/Ca particles were resistant in simulated gastric fluid, but released 70-90% of encapsulated compounds in simulated intestinal fluids while Cru/Cs particles were resistant in both simulated gastric and intestinal fluids and released less than 20% of the compounds. Although the surface of both particles was composed of gastric-resistant cruciferin which was degraded in simulated intestinal fluid, the particles showed different release behavior in simulated intestinal fluid which reveals that the particle cores controlled the release rate of encapsulated compounds. The cellular uptake and trans-cellular transport of Cru/Ca and Cru/Cs particles were also investigated using Caco-2 and Caco-2/HT29 co-culture system. Our results showed that the presence of mucus secreted by HT29 cells, co-cultured with Caco-2, had negligible influence on the uptake and transport of both particles. The uptake of negatively-charged Cru/Ca particles was ~3 times higher than positively-charged Cru/Cs. Whereas digestion in simulated intestinal conditions led to dissociation of Cru/Ca particles, only cruciferin coating was digested from Cru/Cs particle surface; and as a result, the cellular uptake and transport of digested Cru/Cs particles were higher than the undigested form. The presence of mucus in Caco-2/HT29 co-culture decreased the cellular uptake and transport of digested Cru/Cs particles compared to the undigested particles which might be due to the mucoadhesive property of exposed chitosan-based particle core. Energy-dependent mechanism was dominated for uptake of the undigested and digested particles. In the last part of the thesis, the potential chaperone-like activity of napin was evaluated. The results showed that napin had a chaperone-like activity against thermal aggregation of ovotransferrin (OT) in a heat treatment. The chaperone-like activity of napin might be due to formation hydrophobic interactions between napin and OT fibril cores. The interactions decreased β-sheet and random coil structures in the fibril cores while increased α-helix. The results of this thesis supported the potential application of cruciferin for encapsulation of bioactive compounds, and also show a chaperone-like activity for napin. The difference in the application of the proteins was due to their distinct properties; while cruciferin formed particles through aggregation, napin has potential to suppress aggregation of other proteins.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R33B5WG7J
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Agricultural, Food, and Nutritional Science
  • Specialization
    • Food Science and Technology
  • Supervisor / co-supervisor and their department(s)
    • Dr. Jianping Wu, Department of Agricultural,Food and Nutritional Sciences
  • Examining committee members and their departments
    • Committee Chair: Dr. Feral Temelli, Department of Agricultural,Food and Nutritional Sciences
    • Dr. Dr. Larry D. Unsworth, Department of Chemical & Materials Engineering
    • Dr. Loong-Tak Lim, Department of Food Science, University of Guelph
    • Dr. Afsaneh Lavasanifar, Faculty of Pharmacy and Pharmaceutical Sciences
    • Dr. Lingyun Chen, Department of Agricultural,Food and Nutritional Sciences