Carotenoids purification

Patent Data Related to Extraction, Purification, and Formulation of Carotenoid Pigments for Food and Feed Applications... 

Chen, F. et al.. Isolation and purification of the bioactive carotenoid zeaxanthin from the microalga Microcystis aeruginosa by high-speed counter-current chromatography, J. Chromatogr, 1064, 183, 2005. 

Because plants present chlorophylls and carotenoids simultaneously, it may be useful to separate both groups from each other in a laboratory or preparative scale in order to avoid contamination in further purification steps, mainly when they are prepared in large amounts. Clean-up procedures using an open column packed with absorbents such as alumina, magnesia, polyethylene powder, powdered sucrose, DEAE-Sepharose, starch, cellulose, or MgO HyfloSupercel are good approaches. MgO HyfloSupercel in a proportion of 1 1 or 1 2 is the usual adsorbent. Sucrose and cellulose are interesting as they do not alter the chlorophylls, but they are tedious to work with. 

Although saponification was found to be unnecessary for the separation and quantification of carotenoids from leafy vegetables by high performance liquid chromatography (HPLC) or open column chromatography (OCC), saponification is usually employed to clean the extract when subsequent purification steps are required such as for nuclear magnetic resonance (NMR) spectroscopy and production of standards from natural sources. 

Breithaupt, D.E. and Schwack, W., Determination of free and bound carotenoids in paprika (Capsicum annuum L.) by LC/MS, Fur. Food Res. Technol., 211, 52, 2000. Philip, T. and Berry, J.W., A process for the purification of lutein-fatty acid esters from marigold petals, J. Food ScL, 41, 163, 1976. 

Z. E. Jouni and M. Wells, Purification of a carotenoid-binding protein from the midgut of the silkworm, Bombyx mori, Ann. N. Y. Acad. Sci. 691 (1993) 210-212. 

M. R. Lakshman and M. N. Rao, Purification and characterization of cellular carotenoid-binding protein from mammalian liver, Methods Enzymol. 299 (1999) 441 -56. 

Fleischmann, R, K. Studer et al. (2002). Partial purification and kinetic characterization of a carotenoid cleavage enzyme from quince fruit (Cydonia oblonga). J. Agric. Food Chem. 50(6) 1677-1680. 

The purification strategy for CBPs is conceptually simple. Proteins from carotenoid-rich tissues are separated under nondenaturing and relatively aqueous conditions where carotenoids are expected to remain bound to the CBPs. CBPs are then detected by the color of the carotenoids. 

Fujii, H., Matsui, T., Tochihara, S., and Kawaguchi, Y. 1988b. Purification of carotenoids binding protein from larval hemolymph of the yellow blood strain of Bombyx mori. J. Seric. Sci. Jpn., 57(5) 398-404. 

Carotenoids A large number of solvents have been used for extraction of carotenoids from vegetables matrices, such as acetone, tetrahydrofuran, n-hexane, pentane, ethanol, methanol, chloroform [427-431], or solvent mixtures such as dichloromethane/methanol, tetrahydrofuran/methanol, -hexane/acetone, or toluene or ethyl acetate [424,432-435], SPE has been used as an additional purification step by some authors [422,426], Supercritical fluid extraction (SEE) has been widely used, as an alternative method, also adding CO2 modifiers (such as methanol, ethanol, -hexane, water, methylene chloride) to increase extraction efficiency [436-438], In addition, saponification can be carried out, but a loss of the total carotenoid content has been observed and, furthermore, direct solvent extraction has been proved to be a valid alternative [439],... 

Once the carotenoids have been isolated as described in Basic Protocol 1, they can generally be crystallized as an initial step to purification. Actually, what is most likely to happen is a co-crystallization. When working with a nonpolar fraction, a- and (3-carotene may co-crystallize. In the same way, a polar fraction may yield lutein-zeaxanthin crystals. A pure carotenoid product may be obtained by crystallization of a fraction derived from a preparatory chromatographic procedure, which can be done using TLC, HPLC unit F2.3), or in some cases column chromatography. 

The initial wet weight is important as, in many cases, extraction and purification of carotenoids are done to quantify pigment concentrations, which must be related to the fresh material. 

Carotenoids have been of great interest, and their importance in food coloration has been well reviewed by Klaui and Bauernfeind (1981). Extraction, isolation, and purification are described excellently by Schiedt and Liaaen-Jensen (1995). 

Saponification is a purification procedure to remove unwanted lipids and chlorophylls. It has to be omitted when alkali-labile carotenoids (e.g., astaxanthin, fucoxanthin) or carotenoid esters are to be analyzed. To prevent the formation of artifacts produced by aldol condensation between acetone and carotenals, all traces of acetone have to be removed prior to saponification (41). 

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