Improving the bioavailability of collagen-derived peptides: studies in cell culture models

• Author / Creator

  Feng, Mengmeng

• Collagen hydrolysates (CH) are emerging health supplements associated with a growing body of evidence indicating that they may positively influence skin conditions, promote joint and bone health, and even ameliorate hypertension and type 2 diabetes. The biological effects of CH, however, are limited by their low bioavailability. This thesis was committed to improving the bioavailability of CH and collagen-derived peptides, and, interested in elucidating the effects they have on the human dermal fibroblast. The strategy of the first part of the thesis focused on facilitating the absorption of CH by reducing the molecular weight (MW) through enzymatic hydrolysis, as documented in study 1. A practical and efficient method was developed to extract collagen from extremely tough and highly cross-linked bovine hide. This was achieved by a pretreatment with alkaline conditioning, and an extraction process using acetic acid and pepsin. The final product was highly pure and pepsin-acetic acid soluble collagen. The combination of two commercial enzymes, Alcalase and Flavourzyme, was determined to be the best treatment to hydrolyze the collagen generating over 80% of peptides with MW lower than 2 kDa. The in vitro absorption experiment using a human intestinal Caco-2 cell model revealed the positive correlation between a lower MW and a higher transepithelial transport of CH. In addition to the first part which was in an effort to increase the effective proportion of CH that can be absorbed, the second part of the thesis focused on introducing other absorption pathways through chemical modification of collagen-derived peptides, which is summarized in study 2 and study 3. A novel glycopeptide was synthesized from a model collagen peptide Pro-Hyp by conjugating with an amino sugar glucosamine (GlcN). After purification and MW identification, the glycopeptide was clarified to be an amide derivative by nuclear magnetic resonance spectroscopy, i.e. Pro-Hyp-CONH-GlcN. Later, the glycopeptide was subjected to simulated gastrointestinal digestion and Caco-2 cell transport. It showed a desirable enzymatic stability (~90% remaining) with superior permeability compared to its parent peptide Pro-Hyp (Papp 2.82 ×10-6 cm/s vs. 1.45 ×10-6 cm/s). The improvement was achieved by introducing the activity of an extra transport pathway - GLUTs; GlcN moiety in the glycopeptide molecule was structurally comparable to the free GlcN, which makes the glycopetide transported similarly as GlcN. Study 3 investigated effects of the glycopeptide on human dermal fibroblasts, the primary resident cells that account for skin function, and cells mainly affected by health benefits of CH. Interestingly, the glycopeptide activated the proliferation of fibroblasts to 1.5-fold of untreated control. This activity was similar with its parent collagen peptide, so was the performance in stimulating the hyaluronic acid (HA) biosynthesis. The effective concentration of both the collagen peptide and the glycopeptide for the stimulation was revealed to be 200 nmol/ml, representing a physiologicall evel observed in animal studies. The underlying mechanisms were linked to their ability in arresting more cells in the DNA synthesis phase for the cell growth stimulation, and in up-regulating the transcription of hyaluronan synthase genes that encode HA synthesis. This doctoral project provides strategies and a foundation for future research to improve the bioavailability of collagen protein hydrolysates by reducing MW through enzymatic hydrolysis. Also, the chemical modification by glycosylation represents a novel approach to facilitate intestinal absorption of collagen-derived bioactive peptides.

• Subjects / Keywords

  • Skin fibroblast
  • Hyaluronic acid
  • Collagen peptide
  • Bioavailability
  • Glycopeptide
  • Caco-2 cell

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• 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.