Graduate Thesis Or Dissertation
 

Analysis of Intact Glycomacropeptide Derived from Bovine Kappa-casein in Whey Protein Isolate and Intestinal Digesta

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/sf268c389

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  • Kappa-casein glycomacropeptide (CMP), a 64-amino-acid peptide, is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of all the different distinct CMP forms present in whey protein. The full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In the first part of this study, a top-down approach-based analytical method was developed to profile CMP and CMP-derived peptides in one analytical CMP standard, one WPI and two commercial CMP products by using Orbitrap mass spectrometry combined with a nano-liquid chromatography under Electron-Transfer/Higher-Energy Collision Dissociation mode. Fifty-three distinct intact CMP forms were identified in the four samples. In addition to the intact CMPs, 166 distinct CMP-derived peptides were identified in the four samples, likely generated from partial hydrolysis during whey processing or storage. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract where they can exert these functions. In contrast to the analysis of highly purified CMP, identifying the intact and digested CMPs in intestinal digesta samples requires CMP extraction and purification procedures prior to LC-MS/MS acquisition. In the second part of the study, five solid-phase-extraction (SPE) methods, porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), C18, and ion exchange chromatography (anion exchange chromatography and cation exchange chromatography), were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples. CMP does not survive intact to the intestine, and to investigate the bioactivities of CMP in humans has to use the CMP-derived peptides rather than undigested CMP powders. We successfully analyzed CMP in commercial WPI, CMP isolate powder and intestinal digestive samples using C18 and PGC-SPE with a top-down LC-MS/MS- based approach. This study discovered that CMP is mostly in the form of CMP-derived peptides intestinal digesta samples and this result will shape future work as it points to CMP-derived peptides being the key bioactive constituents rather than the intact form.
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  • Pending Publication
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  • 2020-09-03 to 2021-10-03

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