Title page for ETD etd-1113102-150012

Type of Document Master's Thesis
Author Antunes, Tania Serigado
Author's Email Address tantun1@lsu.edu
URN etd-1113102-150012
Title Permeability of Rice Cystatin across Caco-2 Cells
Degree Master of Science (M.S.)
Department Food Science
Advisory Committee
Advisor Name Title
Jack N. Losso Committee Chair
Fred F. Shih Committee Member
Terry H. Walker Committee Member
  • biotechnology
  • protein chemistry
  • purification
  • gastrointestinal tract conditions
Date of Defense 2002-10-30
Availability unrestricted
The present work aimed at recovering, purifying, and testing rice cystatin or oryzacystatin I (OCI) bioavailability, to provide scientific evidence that rice cystatin can be used as a functional food ingredient. OCI was extracted from rice bran using 25 mM sodium phosphate buffer containing 0.15 M NaCl at pH 7.0. The resulting homogenate was heat treated, cooled and precipitated with ammonium sulfate. The recovered protein was dialyzed against 50 mM sodium acetate buffer pH 4.8 and sequentially purified by cation exchange, size exclusion, and anion exchange chromatography. Nine hundred and seventy micrograms of protein were obtained from 1 kg of rice bran. SDS-PAGE provided one pure band. MALDI-MS identified the protein at 11,538 Da. Overall there was a 14-fold increase in specific activity throughout the purification process.

OCI was hydrolyzed with chymotrypsin in 20 mM sodium phosphate buffer pH 7.4 for 2.5 hours at 37C at an enzyme to protein ratio of 1:100. The resulting chymotryptic peptides yielded a higher inhibitory activity compared to unhydrolyzed OCI (145% increase). The permeability of OCI and OCI chymotryptic peptides across Caco-2 cells was evaluated for 3 hours at 37C. Unhydrolyzed OCI did not cross the cell monolayer. OCI chymotryptic peptides were uptaken by the Caco-2 cells, as they were not detected in the apical side of the cells either by MALDI MS or papain inhibitory activity assay. After three-hour incubation, only one peptide with a molecular weight of 5.8 kDa was detected in the basolateral side of the cells. The peptide that crossed the basolateral membrane had no inhibitory activity versus papain. The significance of these findings and future research direction will be discussed.

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