Date of Graduation

5-2013

Document Type

Thesis

Degree Name

Master of Science in Food Science (MS)

Degree Level

Graduate

Department

Food Science

Advisor

Luke Howard

Committee Member

Andy Proctor

Second Committee Member

Neil Allison

Keywords

Biological sciences; Extraction; Flash chromatography; Food science; Hplc analysis; Tocotrienol, Vitamin e

Abstract

The tocols (alpha-, beta-, gamma- and delta- tocopherols (T); alpha-, beta-, gamma- and delta-tocotrienols (T3)) make up the vitamin E family of antioxidants. Tocols have many health benefits. Gamma-T3 has been shown to protect against harmful radiation effects in mice. During rice bran oil refining up to 90% of tocols in rice bran are lost to the deodorizer distillate. The objective of this study was to optimize parameters for analyzing and isolating tocols from rice bran oil deodorizer distillate (RBODD). NP-HPLC analysis of tocol standards was optimized by testing ten mobile phases. Tocol extraction was optimized by testing three concentration solvents (acetonitrile, methanol, ethanol) using various solvent:RBODD ratios (5:1, 10:1, 15:1, 20:1) at two temperatures (4°C, -20°C). Hexane (HX)-ethyl acetate (EA)-acetic acid (AA) (97.3:1.8:0.9 v/v/v) provided baseline resolution of all eight tocols. All interactions including solvent*ratio*temperature had a significant effect on tocol levels in the extracts (p<0.0001). Acetonitrile (10:1, 4°C) was the most efficient solvent with extracts containing 57.4 mg tocopherols/g concentrate and 61.4 mg tocotrienols/g concentrate. These conditions provided extracts with 1.55- and 1.34-fold higher tocopherols and 2.23- and 1.82-fold higher tocotrienols than extracts obtained with ethanol and methanol, respectively. Tocol contents in acetonitrile and ethanol extracts were highest when incubated at 4°C, while tocol content in methanol extracts were highest at -20°C. Under the conditions described above, tocopherol composition was 34.0% alpha-T, 3.4% beta-T, 44.9% gamma-T and 17.4% delta-T and tocotrienol composition was 25.5% alpha-T3, 72.8% gamma-T3 and 1.7% delta-T3. Flash chromatography may be used to isolate a fraction rich in gamma-T3 with a minimal proportion of alpha isomers, which may interfere with radioprotective properties. Use of ~1% load size and silica sample cartridge provided acceptable tocol separation. A mobile phase of 100% HX-EA-AA (97.3:1.8:0.9 v/v) gave a fraction with a concentration of 138.2 mg gamma-T3/g extract (47.0% purity). This purity was significantly lower than the fraction obtained using a linear gradient of 0.8% EA-AA (99.1:0.9 v/v) added to HX-AA (99.1:0.9 v/v), which contained 96.5% gamma-T3 (69.3 mg/g). The ideal gradient system should be chosen according to the final usage of the gamma-T3 fraction.

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