Organic compounds -- part of the class 532-570 series – Organic compounds – Oxygen containing
Reexamination Certificate
2001-10-10
2002-04-30
Shippen, Michael L. (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Oxygen containing
Reexamination Certificate
active
06380442
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
None
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not applicable.
BACKGROUND OF THE INVENTION
The present invention relates to carotenoids that have beneficial effects in humans and more particularly to lutein and its production.
Lutein, (3R,3′R,6′R)-&bgr;,&egr;-carotene-3,3′-diol, has three asymmetric centers, C-3, C-3′, and C-6′. The absolute configuration of lutein in foods and human serum/plasma is known to be 3R,3′R,6′R. In small trace amounts, another configurational isomer of lutein (3R,3′S,6′R) also has been shown to be present in human sera. Of special importance for present purposes is the all-trans isomer or E-isomer of lutein.
In recent years, human and animal studies have established the various beneficial effects of carotenoids, especially lutein and zeaxanthin, in preventing age related macular degeneration and various forms of cancer, due to their antioxidant activity. Fruits and vegetables are the richest sources of a variety of natural carotenoids, of which some are present in only trace amounts. Most of the earlier studies and isolation processes related to carotenoids were targeted towards individual compounds, such as, &bgr;-carotene, lycopene, and lutein. However, recent studies indicate that a mixture of carotenoids may have additional beneficial effects over pure compounds due to synergistic effects. Hence, development of a commercial process for the production of dietary carotenoid mixtures enriched in one or more carotenoids, such as all-trans lutein, may be of importance. These mixtures can be used in nutritional supplements or as food additives.
Although present in green vegetables, yellow/orange fruits and vegetables, marigold flower petals are one of the richest sources of lutein, along with other carotenoids, which occur acylated with fatty acids. The petals are extracted with a lipophilic solvent and the esters are hydrolyzed by saponification to obtain lutein, along with other carotenoids, in free form. Several patents and publications propose the isolation of lutein from marigold petals on a commercial scale. The publications in general focus on isolation of lutein in a pure form and involve multiple process steps.
U.S. Pat. No. 5,382,714 reports that saponified marigold oleoresin from Kemin Industries (Des Moines, Iowa) containing free lutein is the preferred starting material for the isolation of pure lutein. The purification steps involve multiple solvents, cold temperatures, and are very time consuming in commercial production.
U.S. Pat. No. 5,648,567 teaches a process for the isolation of lutein from marigold oleoresin at 74% purity. This process employs propylene glycol and an aqueous alkali to saponify a hexane extract of dried marigold petals containing lutein esters at 70° C. in 10 hours. The process has several disadvantages. For example, the hydrolysis of lutein and zeaxanthin esters in the marigold oleoresin is conducted in an aqueous solution in the presence of propylene glycol, in which the fatty acid esters of lutein and zeaxanthin have very low solubility. As a result, this process requires high temperatures of up to 70° C. and 10 hours to complete the saponification on a commercial scale. This can result in the degradation and unwanted isomerization of lutein and zeaxanthin. Additionally, due to the high viscosity of propylene glycol, the saponified product is continuously subjected to high temperatures ranging from 70° to 85° C. during handling and several purification steps. This unnecessary exposure to heat in the presence of air can result in oxidative degradation of lutein and zeaxanthin with the consequent formation of a number of degradation side products.
U.S. Pat. No. 6,262,284) describes the simultaneous extraction and saponification of carotenoids from marigold dry flower petals. Again, the process has several drawbacks. Marigold petals contain a maximum of 1-2% total carotenoids. The dry petals are extracted using tetrahydrofuran at a 1:10 dry matter to solvent ratio. The extract is used for saponification without further concentration, which results in the use of large volumes of solvent during commercial production. The solvent also is unstable and produces peroxides, which may degrade the carotenoids. The low levels of carotenoids in the meal combined with the high solvent to meal ratio and the final recrystallization step make commercial viability of the process doubtful.
BRIEF SUMMARY OF THE INVENTION
The present invention is a convenient and effective process to isolate carotenoids, especially lutein, from marigold oleoresin (as the preferred source) with minimal use of organic solvents. The process involves hydrolysis of the carotenoid esters in marigold oleoresin using isopropanol, water, and alkali, for a minimum of 60 to 90 minutes at a temperature of about 60° to 65° C. The hydrolyzed carotenoids are precipitated from the saponified mixture using water. The precipitate is recovered by centrifugation, followed by purification of the precipitate with repeated water washings, and a drying step to obtain a fine crystalline material.
The process can be used to recover the carotenoids from oleoresins or extracts containing low levels of carotenoids, a mixture of oleoresins with different carotenoid profiles, and oleoresins obtained by supercritical extraction. In the case of plant extracts containing the carotenoids in a free form, such as, for example, spinach, the saponification step separates the carotenoids from chlorophyll and other contaminants.
Advantages of the present invention include, inter alia, hydrolysis of the esters can be efficiently completed at a lower temperature and in a shorter duration of time, because the oleoresin is completely soluble in isopropanol at a temperature of about 60° to 65° C. Another advantage is that the remaining process steps are carried out at room temperature, thus reducing the risk of oxidative degradation of carotenoids. As further advantage, the process recovers even the trace amount of other carotenoids present in the oleoresin in addition to the recovery of major carotenoids. Yet further advantages are that, the process is less time consuming, economical, and commercially feasible compared to prior processes, because the invention does not involve use of multiple solvent extractions or recrystallization steps. These and other advantages will become readily apparent to those skilled in the art based on the disclosure set forth herein.
DETAILED DESCRIPTION OF THE INVENTION
The invention is the isolation of carotenoid mixtures containing high levels of desired specific compounds from plant extracts known to synthesize the desired compounds at high concentrations. Specifically, commercially available food grade marigold oleoresin produced by hexane extraction can be used as the starting material for the isolation of an all-trans lutein enriched product. Marigold flower (Tagetes sp., such as
Tagetes erecta
) is reputed to be the best possible commercial source for all-trans lutein as it contains lutein mono and diesters as the major carotenoid constituents. Marigold oleoresin obtained from the dry flower petals contains around 5% to 20% lutein esters, based on the cultivar and the extraction process. In addition to lutein, marigold oleoresin also contains all-trans zeaxanthin, &agr;- and &bgr;-cryptoxanthin, &bgr;-carotene, and traces of other carotenoids.
In the present invention, the oleoresin is dissolved in food grade isopropanol to form a free flowing solution at a temperature within the range of from about 60° to 65° C. In a typical process, one weight part of the oleoresin is dissolved in excess (e.g., 2-3 volume parts) of the solvent. The impurities present in the oleoresin, such as, waxes, resins, and non-carotenoid pigments, also are soluble in isopropanol.
An aqueous 50% potassium hydroxide solution, for example, is added to the solution under constant agitation. The amount of the alkali required is approximately 1.5 to 2 times the concentration of the total
Kagan Daniel I.
Madhavi Doddabele L.
BioActives, LLC
Mueller and Smith LPA
Shippen Michael L.
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