Carotenoid extracts

Carotenoids are probably the best known of the colorants and certainly the largest group of pigments produced in nature with an annual production estimated at 100,000,000 tons. Most of this is fucoxanthin produced by algae in the ocean and the three main pigments, lutein, violaxanthin and neoxanthin in green leaves.10 Over 600 carotenoid compounds have been reported. 

The chemical structure of some typical carotenoids is shown in Fig. 8.2. Beta-carotene occurs in nature usually associated with a number of chemically closely related pigments and extracts have been used as food colorants for many years. For example, palm oil has a high concentration of carotenoid pigments, primarily beta-carotene and about 20 others. Cmde palm oil has been used extensively as a cooking oil because of its desirable flavor and as a general 

There is little toxicological data available for extracts of carrots, alfalfa, com oil, palm oil, tomatoes, etc. The JECFA had no objections to their use as food colorants provided that the levels of use did not exceed that normally present in vegetables. A number of toxicity experiments were conducted on Dimaliella algae in view of its increasing importance in the health food area. Twelve studies on D. salina indicated no problems. ( is beta-carotene was absorbed to a lesser extent than trans beta-carotene. Furahashi suggested a no-observed-effect level (NOEL) of 2.5 g kg/day for extracts from D. Hardawil 2 The Joint Expert Committee on Food Additives of the World Health Organization/United Nations (JECFA) did not establish an NOEL or an ADI because of the variation in the composition of the products. 

Carotenoids are of physiological interest because some of them are precursors of vitamin A. They have been in the news recently because many exhibit radical or single oxygen trapping ability and as such have potential antioxidant activity in vivo. They may reduce the risk of cardiovascular disease, lung cancer, cervical 

The dramatic increase in the health aspects of the carotenoids has spurred a great deal of interest in these compounds as colorants. The prospect of having both a health and a colorant aspect is very appealing to merchandisers so we can expect an increase in the number of carotenoid extracts available. But with over 600 carotenoids existing in nature, it will be difficult to determine which compounds exhibit health effects. 

---

In milk approximately 90% of the yellow color is because of the presence of -carotene, a fat-soluble carotenoid extracted from feed by cows. Summer milk is more yellow than winter milk because cows grazing on lush green pastures in the spring and summer months consume much higher levels of carotenoids than do cows ham-fed on hay and grain in the fall and winter. Various breeds of cows and even individual animals differ in the efficiency with which they extract -carotene from feed and in the degree to which they convert it into colorless vitamin A. The differences in the color of milk are more obvious in products made from milk fat, since here the yellow color is concentrated. Thus, unless standardized through the addition of colorant, products like butter and cheese show a wide variation in shade and in many cases appear unsatisfactory to the consumer. 

Preparation of carotenoid extract from plant oleoresin and hydrolysis with alkahne reagent in polar organic solvents (ether, polyhydroxyl alcohol, and ether alcohol)... 

Mapari, S. A.S. et al., Exploring fungal biodiversity for the production of water-soluble pigments as potential natural food colorants, Curr. Opin. BiotechnoL, 16, 231, 2005. Dufosse, L., Mabon, R, and Binet, A., Assessment of the coloring strength of Brevi-bacterium linens strains spectrocolorimetry versus total carotenoid extraction/quan-tification, J. Dairy ScL, 84, 354, 2001. 

In order to obtain reliable results, three steps are involved in the analysis sampling and sample preparation carotenoid extraction, separation, identification. 

The carotenoid extract obtained by extraction of fresh food with a water-soluble solvent contains large amounts of water from the sample. In order to remove the water and solvent, in the case of acetone, carotenoids are transferred to petroleum ether, diethyl ether, or a mixture by adding small portions of the solvent extract and a large amount of water in a separatory funnel. The remaining traces of water can be removed either by addition of anhydrous Na2S04 or ethanol to form the azeotropic mixture. 

A general procedure that our laboratory generally employs is the addition of an equal amount of methanolic 10% potassium hydroxyde (KOH) to an ethereal carotenoid extract. This solution is bubbled with N2 and allowed to stand overnight at room temperature. Other conditions that shorten time at room temperature have also been used, such as saponification of the dichloromethane (CH2CI2) extract with the same amount of 10% KOH in MeOH for 1 hr (peppers and fruits ) and ethereal extract treated with 30% methanolic KOH under N2 for 3 hr (green leaves, vegetables and fruits ). 

An open column packed with neutral aluminium oxide (grade III) slurry is generally used for semi-preparative separation of large amounts of carotenoid extract, revealing three broad bands (1) carotenes and epoxy-carotenes constitute the first fraction to elute with petroleum ether, (2) monohydroxy and keto-carotenoids with 50 to 80% diethyl ether in petroleum ether are next, and (3) finally, the polyhydroxy carotenoids elute with 2 to 5% diethyl ether in ethanol or... 

Chromatography may be necessary to separate some carotenoids. A saponified carotenoid extract from carrots was applied to OCC on MgO and two fractions were eluted. Successive crystalhzation with petroleum ether and MeOH was carried out to obtain a- and P-carotene crystals, with 99 and 98% purity levels, respectively. ... 

Lutein is a major component of many plants. It is a component of most of the carotenoid extracts suggested as food colorants. 

The success of the carotenoid extracts led to the commercialization of synthetic carotenoids, some with the same chemical structure as those in the plant extracts and others with modifications to improve their technological properties. The yellow beta-carotene was synthesized in 1950, followed by the orange beta-8-carotenal in 1962 and the red canthaxanthin in 1964. A number of others soon followed, methyl and ethyl esters of carotenoic acid, citraxanthin, zeaxanthin, astaxanthin, and recently lutein. 

Fig. 2.24. C30 chromatograms of carotenoids extracted from human serum (a) xanthophylls fraction, 7 93 (v/v) MTBE-methanol mobile phase (b) a- and / -carotenes fraction, 11 89 (v/v) MTBE-methanol mobile phase (c) lycopene fraction, 38 62 (v/v) MTBE-methanol mobile phase. Tentative peak identifications (a) 1, 13-c/s-lu- lutein 2, 13 r/.vlutein 3, a//-/ra s-lutein 4, zeaan-thin 5-7, unidentified P,e-carotenoids and 8, / -cyrptoanthin (b) 1-2, unidentified ae-carotene isomers 3, 15-eH -/f-carotenc 4, 13-cw-/ -carotene 5, all-trans-a-carotene 6, all-trans-P-carotene and 7, 9-ci.v-/3-carotene and (c) 1-11 and 13, c/s-lycopene isomers and 12, all-trans-lycopene. Reprinted with permission from C. Emenhiser el al. [51].

In addition to the absorption maxima of the carotenoids, the shape of the spectra provides important information for identification of purified carotenoid extracts or pure standard (while the identity of the standard is generally not in question, it is a good idea to check the purity by fine structure analysis). Fine structure... 

Fig. 11 Chromatogram of chlorophylls, chlorophyll derivatives, and carotenoids extracted from spinach leaves and analyzed by HPLC with a photodiode array detector. Chd = chlorophyllide, Pb = pheophorbide, Chi = chlorophyll, Po = pheophytin. (From Ref. 99.)...

Pumpkin, tunicate, oyster Epoxy carotenoids Extraction with acetone, partitioning, fractioned with silica, purified with HPLC using C18 Chloroform- ACN MS/MS/FAB(+) 80... 

Saponification is often used to extract xanthophylls as well as remove chlorophylls and lipids from samples prior to analysis, as these compounds can interfere with the chromatographic detection. Although saponification with methanol and potassium hydroxide is routinely used to facilitate carotenoid extraction, numerous studies indicate that saponification can also result in losses of carotenoids. For example, Khachik et al.60 demonstrated that saponification actually caused the loss of total carotenoids in samples. Alternatively, enzymatic saponification using lipase can be used to help prevent the loss and isomerization of some carotenoids. Fang et al.32 suggested that saponification of plasma samples should be avoided to prevent unnecessary lycopene degradation. 

Seaweed lipid/carotenoid extraction, analysis of total lipid, lipid... 

Seaweed Lipid/Carotenoid Extraction, Analysis of Total Lipid, Lipid Classes and Fatty Acids... 

Machmudah, S. Kawahito, Y. Sasaki, M. Goto, M. 2008. Process optimization and extraction rate analysis of carotenoids extraction from rosehip fruit using supercritical CO2. J. Supercrit. Fluids 44 308-314. 

---