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 37°C 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 37°C. 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.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-1113102-150012 |
| Date | 14 November 2002 |
| Creators | Antunes, Tania Serigado |
| Contributors | Fred F. Shih, Jack N. Losso, Terry H. Walker |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-1113102-150012/ |
| Rights | unrestricted, I hereby grant to LSU or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University Libraries in all forms of media, now or hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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