Separation and purification of nootkatone from fermentation broth of Yarrowia lipolytica with high-speed counter-current chromatography

Abstract

Nootkatone is an important functional sesquiterpene, which can be obtained by the biotransformation of valencene. It is increasingly important because of its pleasant citrus aroma and physiological effects. Yarrowia lipolytica is beneficial for biotechnology applications and has ability to transform valencene to nootkatone. High-speed counter-current chromatography (HSCCC) was used to isolate and purify the product of nootkatone in this study. The suitable two-phase solvent system was selected and the optimum separation conditions were determined. The partition coefficients of nootkatone and the separation factor between nootkatone and valencene were considered as the indexes. The results showed that there were numerous products during the transformation of valencene by Yarrowia lipolytica, and the content of nootkatone was 13.75%. The obtained nootkatone was separated by HSCCC with a solvent system n-hexane/methanol/water (5/4/1, v/v). The final purity of nootkatone was 91.61 ± 0.20% and the elution time was 290-310 min. The structure of nootkatone was identified by gas chromatography-mass spectrometry (GC-MS), infrared spectrum and nuclear magnetic resonance hydrogen spectroscopy (¹H NMR). This was the first report on the separation of nootkatone from the fermentation broth by HSCCC. This study proved that HSCCC could be used as an effective method to separate and purify the nootkatone from valencene transformed by Yarrowia lipolytica with n-hexane/methanol/water (5/4/1, v/v).

Keywords: Biotransformation; High-speed counter-current chromatography; Nootkatone; Purification; Valencene; Yarrowia lipolytica.

Fig. 1

HSCCC chromatogram of target compound separation with different two-phase solvent systems. A n-hexane/dichloromethane/acetonitrile/ethyl acetate (6/1/4/1, v/v); B n-hexane/ethyl acetate/methanol/water (5/1/4/1, v/v); C n-hexane/ethyl acetate/methanol/water (5/0/4/1, v/v)

Fig. 2

A SPME–GC–MS detection of total ion flow chart of conversion products in fermentation broth. B GC–MS diagram of products obtained from elution and separation of n-hexane/ethyl acetate/methanol/water (5/0/4/1, v/v). 1, valencene; 2, nootkatone

Fig. 3

A Mass spectrogram of nootkatone from separation and purification of products with n-hexane/ethyl acetate/methanol/water (5/0/4/1, v/v). B Infrared chromatographic analysis of standards and separated products with different solvent systems. (a) nootkatone standard; (b) valencene standard; (c), product separated with n-hexane/ethyl acetate/methanol/water (5/0/4/1, v/v); (d), product separated with n-hexane/ethyl acetate/methanol/water (5/1/4/1, v/v); (e), product separated with n-hexane/dichloromethane/acetonitrile (10/1/10, v/v); f, product separated with n-hexane/acetonitrile/chloroform (6/5/2, /v/v); g, product separated with n-hexane/dichloromethane/acetonitrile/ethyl acetate (6/1/4/1, v/v). C ¹H NMR of nootkatone from separation and purification of products with n-hexane/ethyl acetate/methanol/water (5/0/4/1, v/v)