Preparation and Characterization of Immunomodulatory Peptides from Spent Hen Muscle Proteins

• Author / Creator

  Yu, Wenlin

• Each year the egg industry produces about 18 million spent hens in Canada. Since a viable market is not available, spent hens are a liability to the egg producers who have to pay the cost associated with disposal. The overall objective of this thesis was to prepare and characterize bioactive peptides with immunomodulatory properties from underutilized spent hens. We hypothesized that spent hens could be a good source of muscle proteins for preparing immunomodulatory peptides. Spent hen muscle proteins were hydrolyzed by nine proteases with degree of hydrolysis and the nitrogen recovery ranging from 11.2 to 32.7%, and from 44.4 to 86.5%, respectively; the molecular weight of all hydrolysates was less than 12kDa. In LPS-activated macrophage-like U937 cells, at a concentration of 500 µg/mL, Protease M and Protex 50FP hydrolysates inhibited the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α by 13% and 25%, 15% and 4%, respectively, in comparison to 28% and 57% inhibition by lactoferrin. The immunomodulatory properties of these two hydrolysates were further studied in healthy weanling Sprague-Dawley rats. Feeding at a dose of 5% (w/w diet based), only Protex 50FP hydrolysate exhibited a stimulatory effect on IL-10 (regulatory cytokine) in splenocytes stimulated ex vivo with mitogens (lipopolysaccharides, LPS; pokeweed mitogen, PWM; p<0.05). Meanwhile, no detrimental effect was observed in the young rodents at this dosage. The IL-10 stimulatory activity and in vitro IL-6 inhibitory activity suggests potential anti-inflammatory properties of this hydrolysate, which prompted us to test the hydrolysate in an inflammation-related disease model. In a hapten-induced colitis model, three weeks pretreatment of Protex 50FP hydrolysate attenuated the weight loss up to 48 hr post disease induction. On day 5 after colitis treatment, this hydrolysate did not show any protective effect on colon damage and immune functions; but changes were observed on phenotype, including a higher proportion of Th cells expressing CD25 (IL-2 receptor) in spleen and a lower proportion of T cells expressing CTLA-4 (activation marker) in mesenteric lymph nodes. To characterize the peptides with IL-6 inhibitory activity, these two hydrolysates (Protex 50FP and Protease M) were subjected to ultrafiltration, cation exchange chromatography and reverse-phase chromatography. Twenty five novel peptide sequences were identified from these two hydrolysates. This thesis reported for the first time the preparation and characterization of immunomodulatory peptides from spent hen muscle proteins. This study might lead to alternative method of spent hen uses for preparing “value-added” functional bioactive peptides.

• Subjects / Keywords

  • Immunomodulatory peptides
  • Characterisation
  • Preparation
  • Spent hens

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3S46HF15

• 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.