MXPA05010406A - Method for extracting lutein from green plant materials - Google Patents

Abstract

The present invention provides a method for extracting carotenoids from green plant materials using supercritical fluid extraction. A first and second supercritical fluid extraction is performed on the green plant composition at two different pressures to obtain two extracts. The first extract includes substantial amounts of B-carotene. The second extract may have a controlled concentration of B-carotene, and includes substantial amounts of lutein.

Description

METHOD TO REMOVE THE LUTEINE OF GREEN VEGETABLE MATERIALS FIELD OF THE INVENTION The present invention generally concerns the field of extraction of natural products. More particularly, it concerns the use of supercritical fluid for the extraction of carotenoids from green plant materials.

BACKGROUND OF THE INVENTION Carotenoids are highly colored materials that are found naturally, which are widely distributed in nature. Carotenoids can be classified as hydrocarbon carotenes or antoxanthines, which are oxygenated derivatives of carotenes. Representative examples of the carotenes include β-carotene, alpha-carotene, and lycopene. Examples of antoxanthins include lutein, astaxanthin, canthaxanthin, zeaxanthin, and capsorubin. It has been shown that carotenoids have antioxidant properties and have been studied for the prevention of cancer and other human diseases. Carotenoids are naturally present in leaves, flowers, and edible fruits and are easily obtained from flowers (ie calendula), strawberries, and root tissues (ie, carrots). Hydrocarbon carotenes, such as β-carotene and lycopene, are typically present in an uncombined free form, which is trapped in chloroplast bodies in plant cells. Antoxanthins such as lutein are abundant in numerous yellow or orange fruits and vegetables such as alfalfa, spinach, cabbage, and leafy green plant materials. The free form of carotenoids provides better absorption when consumed in food or as a supplement. Lutein is an antoxanthin found in high concentrations in the macula of the eye and in the central part of the retina. It plays important roles in vision to help filter ultraviolet wavelengths of light to prevent damage to the lens and macula of the eye. It is believed that the antioxidant properties of lutein help to protect the macula, which is rich in polyunsaturated fats, from free radicals induced by light. Lutein can not be produced by the body, and consequently, must be ingested. Accordingly, lutein has become increasingly used in nutritional supplements for the prevention and / or treatment of vision loss due to macular degeneration, cataracts and retinitis pigmentosa. Lutein has been shown to have significant potential in the prevention of age-related macular degeneration (AMD), which causes irreversible blindness among Americans 65 years of age or older. Lutein helps build the density of macular pigment, a critical factor in the health of the macula and the retina. It was found that high intake of green vegetables rich in lutein (spinach and cabbage) reduced the speed of AMD by 40%, while beta-carotene, vitamin A, and vitamin E, did not appear to have an effect (Seddon et al. , 1994). It has been shown that the accumulation of lutein in the macular pigment is dependent on dietary intake and that the density of the macular pigment is related to the preservation of visual sensitivity and protection of AMD (Pratt, 1999, Richer, 2001). Other problems of loss of vision, such as cataracts and retinitis pigmentosa can also be stopped or reduced with a high intake of lutein. The most common source of extracted lutein is from the petals of calendula flowers, which contains one of the highest known levels of lutein and has a low concentration of other carotenoids. Purification methods of fatty acid esters of lutein from marigold flower petals are reported in U.S. Pat. Nos. 4,048,203, 5,382,714 and 5,648,564, in which petals of crushed and dried calendula flowers are extracted with a hydrocarbon solvent. In U.S. Pat. No. 5,648,564, the extraction is carried out 8-10 times with a 60 minute impregnation in hexane solvent for the extraction of the carotenoid from the calendula, and use 320 - 400 liters of hexane per kilo of dry calendula flower petals. The solvent is removed and the residue is dissolved in a hot alcohol. The solution is then filtered and then the lutein fatty acid ester is separated by precipitation. To obtain a more digestible form of lutein extracted from calendula flower petals, the extract is saponified at high pH (10+) or hydrolysates to convert the product to a lutein in free form. The U.S. Patent No. 5,382,714, the use of commercially available saponified calendula oleoresins to crystallize lutein after saponification of oleoresins using organic solvents. The purification of lutein from saponified calendula oleoresins without the use of added organic solvents is reported in U.S. Pat. No.5,648,564. There are several drawbacks to the extraction methods reported previously. For example, the method reported in U.S. Pat. No. 5,648,564 uses caustic products, high pH conditions that can be hazardous and can cause yield losses and vapor exposure, as well as produce toxic waste materials that need to be discarded when finished. Trace amounts of these toxic chemicals and solvents may be present in the final products, which may be a problem for the use of the resulting lutein extract for human consumption. The method reported in U.S. Pat. No. 5,382,714 uses organic and caustic solvents such as hexane, propane diol, and potassium hydroxide for the extraction and saponification processes, which can not be easily removed during the purification process. In addition, no method uses an initial material in which lutein is obtained in its free form. As noted above, carotenoids in free form such as lutein may provide better adsorption in the body during consumption. Therefore, it would be desirable to provide a lutein extraction method that isolates lutein in free form without requiring the use of organic solvents during any step, after extraction of lutein from raw materials for the production of free lutein, for consumption . Lutein is abundantly present in an unesterified form, free in green vegetables such as alfalfa, broccoli, green beans, green pea, lima bean, cabbage, cabbage, spinach, kale, green mustard, green turnips, kiwi, and molasses. Green vegetables can also be rich in a variety of additional nutrients. For example, alfalfa is rich in proteins, minerals and vitamins. It contains the 21 amino acids, and has significant concentrations of vitamins A, D, E, B-6, and K, calcium, magnesium, chlorophyll, linoleic and linolenic fatty acids, phytoestrogens, phosphorus, iron, potassium, trace minerals and various enzymes digestive They also contain several saponins, many sterols, flavonoids, coumarins, alkaloids, acids, additional vitamins, amino acids, natural sugars, proteins, (25% by weight) minerals, trace elements and other essential nutrients. The extraction of lutein from green vegetables can be beneficial because it eliminates the need for the additional chemical step of saponification or spherical fragmentation to liberate free lutein, which is the desired form for better absorption when consumed. However, the isolation and purification of lutein from vegetables has not been economical in the past because time-consuming and costly purification steps have been required to separate lutein from the large amounts of other compounds present in the materials vegetables. Supercritical fluids (SCF), which are gases above their critical pressure and temperature, have been used in certain industries to carry out extractions. SCFs are dense gases in a separate phase, which is different from the normal gas phase. The SCFs have a solvation density and power similar to that of a liquid and diffusion speeds similar to those of a gas. Supercritical fluids are different liquids because their solvent power is very sensitive to pressure changes and can vary over large limits when changing pressure. Extraction by means of SCF offers a relatively quick, simple and low-cost technique for carrying out purification or compound preparations. The majority of the compounds, once dissolved, can be precipitated quickly and cleanly or eliminated from the supercritical fluids by abatement of the pressure and / or temperature or both, to achieve separation. Because a slight change in the pressure or temperature of a system causes significant changes in solubility, the use of SCF facilitates a very efficient isolation procedure for the components that are to be extracted. Using the post-extraction fractionation method with a column designed to allow pressure and temperature drops at different levels to achieve the desired results, further concentration and purification can be carried out. A method of extracting carotenoids such as lutein from alfalfa without using toxic solvents, is reported in Favaty et al., Supercritical CO2 Extraction of Carotene and Lutein from Leaf Protein Concentrates (1988). In the method reported in Favaty, extracts containing mixtures of free lutein and ß-carotene were obtained from alfalfa by means of supercritical extraction in a single-stage extractor. This laboratory-scale extraction was done in a single step, a mixture of lutein, carotene, and other components was extracted from a leaf protein concentrate, with the relative concentrations of the two carotenoids dependent on the extraction pressure used. The carotenoid content obtained from the procedure was 1.5% of the total extract. Although the supercritical extraction method reported in Favaty et al. Overcomes the above problems with safety and health hazards from extractions with conventional solvents, the resulting extracts include an uncontrolled mixture of lutein and other carotenoids in a single extraction. However, given the beneficial health effects of lutein, it would be desirable to obtain an isolated lutein extract containing a substantial concentration of lutein as long as it is substantially free of other carotenoids. It may also be beneficial to obtain extracts with controlled concentrations of lutein and other desired nutrients such as β-carotene and / or fatty acids in order to treat patients with variable nutritional needs based on age (eg, adults versus children) and / or the existence of ocular conditions such as macular degeneration.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides a method for isolating lutein from green plant materials, in which a first supercritical fluid extraction of the green plant material is effected at a first pressure to obtain a first extract. A second extraction by supercritical fluid of the green plant material is then effected at a second pressure to obtain a second extract. The second extract includes lutein, but is substantially free of carotenes such as β-carotene. The second extract of the supercritical fluid used to effect the first and second supercritical extractions is then separated. The first extract can be separated from the supercritical fluid in a similar manner. A variety of green plant materials may be used as the starting material in the method of the present invention. Suitable green materials may include alfalfa, triguillo, barley grass, broccoli, cauliflower, spinach, cabbage, soybeans, green beans, green mustard, green turnips, kale, and green pea. In one embodiment, alfalfa is provided as the green plant material. During the first and second supercritical extraction of the green plant material, the first and second pressures may be from about 8 MPa to about 200 MPa, more particularly from about 10 MPa to about 120 MPa. In one embodiment, the first pressure is lower than the second pressure. For example, the first pressure may be between about 10 and about 40 MPa, and the second pressure may be between about 41 and about 80 MPa. More particularly, the first pressure may be about 20 MPa and the second pressure may be about 65 MPa. The temperature during supercritical extractions may be between about 31 ° C to about 200 ° C, more particularly between about 31 ° C and about 40 ° C, or even more particularly about 35 ° C. The temperature may vary or remain constant during extractions. By optimizing the temperature and pressure at which the first and second supercritical extractions were made, each extract may contain a substantial concentration of a particular substance, such as a desired carotenoid. In one embodiment, the first extract includes a substantial amount of β-carotenes and the second extract includes a substantial amount of lutein, but is substantially free of β-carotene. In another embodiment, the first supercritical extraction is carried out until the green plant material is substantially free of ß-carotenes. Additional extractions may also be carried out at additional pressures and / or temperatures.

After performing the second supercritical extraction, the second extract can be separated from the supercritical fluid by abatement of the second extraction pressure so that the lutein is separated by precipitation of the second extract and on a desired carrier. The first extraction can be separated in a similar way. In one embodiment, the pressure of the first extract can be lowered to approximately 10 MPa and the pressure of the second extract can be lowered to approximately 40 MPa. The first and / or the second extract can then be processed to form a final product suitable for consumption. In another embodiment, the present invention provides a continuous method for obtaining a plurality of extracts of green plant material. A plurality of supercritical extractions can be made at a plurality of pressures to obtain a plurality of extracts. For example, one of the extracts may contain substantial amounts of lutein. Another extract may contain substantial amounts of carotene. Other extracts may contain fatty acids, antoxanthin, zeaxanthin, astaxanthin, canthaxanthin, capsorubin and cryptoxanthin. Said extracts can be obtained by optimizing the pressure and / or the ambient temperature at which the extract is obtained to provide an extract having a substantial concentration of the desired substance. In yet another modality, the present invention provides a method for obtaining lutein, in which a first supercritical extraction is effected at a first pressure and temperature to obtain a first extract. A second supercritical extraction is then carried out at a second pressure and temperature to obtain a second extract. The second extract has a higher concentration of lutein than the first extract. The second extract may additionally include controlled concentrations of β-carotenes and / or fatty acids. In a further embodiment, the present invention provides a method for obtaining a plurality of extracts of green plant material. A first extraction by supercritical fluid is carried out at a first pressure and temperature to obtain a first extract. At least one first extraction of supercritical fluid is then effected at at least one additional pressure and temperature. At least one of the additional extracts includes a higher concentration of lutein than the first extract. At least one of the additional extracts may also include a controlled mixture or combination of desired nutrients. For example, one of the plurality of extracts may include a mixture of lutein, carotene and / or fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a flow chart for the fractionation and extraction of lutein according to one embodiment of the present invention. Figure 2 illustrates a lutein extraction chamber and a flow diagram of the collection system method according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method of extracting carotenoids such as lutein from green plant materials using a supercritical extraction process. The procedure is optimized for pressure and / or temperature during extraction to obtain the maximum concentration of the desired carotenoids. The green plant material used as the initial material for the extraction of supercritical fluid can be derived from any suitable green vegetable, including alfalfa, triguillo. barley grass, broccoli, cauliflower, spinach, cabbage, soybeans, green beans, green mustard, green turnips, kale, and green peas. Although green vegetables can be used in any form (eg wet or dry) that include and retain the desired nutrients for supercritical extraction, a fraction rich in dry or moist chloroplasts of a green vegetable may be particularly useful for the extraction of carotenoids. The fraction rich in chloroplasts can be separated from other plant fractions by means of a process that includes the use of heat, acids, centrifugation, electric field, or flocculants. The fraction rich in chloroplasts can be dried to 5-50% moisture with hot air, infrared heat, microwave radiation or a vacuum oven before extraction with supercritical fluid to preserve the desired components. Before extraction by supercritical fluid, the fraction rich in chloroplasts can be washed with an aqueous solution. This washing step can remove bitter flavors from the chloroplast-rich fraction to provide a more palatable fraction for use in a nutritional supplement. Alternatively, the starting material may be dried in a manner such that the desired nutrients are preserved for subsequent supercritical extraction. Additionally, in this embodiment, the green plant material can be dried in the absence of oxygen if the desired nutrient is sensitive to oxidation by air or oxygen.

Pre-extraction Processing of Green Vegetable Material The green plant material can be processed in a variety of ways before performing supercritical extraction to obtain a desired initial material. In one embodiment, the green plant material is subjected to the wet fractionation procedure illustrated in Figure 1. In this embodiment, the pre-flowering alfalfa can be harvested with standard farm equipment and then cut or crushed into lengths of 1.25 cm. . (/ inch) to 10.16 cm (4 inches). This cutting or grinding process is generally carried out in 1 hour after harvesting to preserve the desired compounds. Cut or crushed alfalfa can be sifted or macerated with rollers or with hammer mill devices that break the plant cell walls. The macerated green crop can then be compressed into a suitable compression device, screw press, or other press that separates the juices from the green vegetables from the fibrous plant material. The residual fibrous plant fraction, or the moist fiber fraction of alfalfa, typically have 55-65% moisture, 14-18% protein, and has most of the nutritional value typical of green forages. This fraction can be used to feed ruminants to dairy or meat cows in either the wet or dry form. Green vegetable juice is a mixture of cellular sap materials, which include water, salts, chloroplasts, and cytoplasmic proteins, enzymes, and cellular compounds. The juice can be further treated by one of several methods to separate the desired components. In a modality, the juice is typically subjected to thermal coagulation at 60 ° C for the chloroplast fraction and at 85 ° C for the cytoplasmic fraction. Alternatively, the juice can be treated by means of acid precipitation, by densities separations in the centrifugal field, or by direct electric current fields. These techniques produce three general fractions: (a) a fraction of green protein chloroplasts; (b) a white cytoplasmic protein fraction; and (c) a fraction of brown juice. In one embodiment, the separation of the green protein concentrates from the brown juice is carried out by centrifugation or filtration methods. In one embodiment, the fraction of green protein chloroplasts of alfalfa is the initial green plant material for the supercritical extraction of lutein from green vegetables using the supercritical fluid. This fraction is rich in plant chloroplasts and is typically composed of 50-55% proteins on a dry basis and has 1.8 to 3.5 g of antoxanthin per kg. The fraction of green chloroplasts can be used wet or can be dried before extraction of carotenoids. The dry form can produce a more stable material for extraction. The fractions of the green vegetable juice can be dried under mild conditions to preserve the desired components. Drying can be accomplished with hot air or other hot inert gases, infrared heat, microwaves, vacuum furnace devices, or any other method or combination of methods to remove water at the desired level. Washing the fraction of green protein chloroplasts with an aqueous solution or water immediately before supercritical extraction may be advantageous. The washing process can eliminate bitter flavors and aromas from the concentrated protein fraction and can make the extract more palatable for subsequent human consumption. Lutein has very little solubility in water, so washing with water causes only minor product losses. This washing step can be particularly beneficial if the post-extraction green protein chloroplast fraction is used as part of a nutritional supplement. Although alfalfa is used as the initial plant material in the reported mode, any recent green crop can be used by wet fractionation, including triguillo, barley grass, broccoli, cauliflower, spinach, cabbage, soybeans, green beans, green mustard, green turnips, kale, and green pea. For example, the wet fractionation procedure reported above can be easily adapted for triguillo and barley grass. Since wet fractionation is similar for alfalfa and herbs, the procedure is the same for most fresh vegetables.

Supercritical Extraction Procedure Once a suitable green material is obtained, supercritical extraction can be carried out by passing supercritical fluids (SCF) through the green plant material. The supercritical fluid used in the method of the present invention may include CO2, CH2CH2, CH3CH3, N2O or other suitable supercritical fluids. A co-solvent may be used in conjunction with the supercritical fluid to increase the solvation power for polar analytes that do not readily dissolve in supercritical fluids. The co-solvents are often referred to as entrainers or modifiers, and are typically a liquid organic solvent such as methanol, ethanol, propylene carbonate, acetone, tetrahydrofuran, formic acid, propylene glycol, or ethyl acetate which are mixed with the dioxide. of carbon. With a trawler, the solvent system has a much higher polarity and is able to solubilize more polar analytes for extraction. It has been shown that the entrainers substantially increase the solubility of zeaxanthin in supercritical carbon dioxide as reported, in part, in U.S. Pat.

No. 5,747,544. In one embodiment the SCF includes ethanol as a tracer at 1-5% concentration in the extracted material. This entrainer can produce better extraction at lower pressures. In one embodiment, the SCF is carbon dioxide, which has a critical pressure of 1070 psi (approximately 7.4 MPa) and a critical temperature of 31 ° C. The power of solvation increases when the pressure and temperature rise above the critical pressure and temperature. The supercritical CO2 can be manipulated at room temperature, making the handling of vulnerable substances easy and safe heat. The danger of explosion and fire associated with large-scale extractions using organic solvents are eliminated with this solvent. In practice, the fluid is passed through green plant materials inside an extraction vessel. The fluid diffuses into the pores of the green plant material matrix, solubilizes the extracts (eg, lutein or carotene) of interest, and then conducts the extracts from the green plant matrix in a solution. The extract is then collected and the green plant matrix (now without the extract) is removed from the extraction vessel. Supercritical fluids have diffusion and favorable viscosity coefficients that provide good mass transfer characteristics. The change in pressure and temperature of the fluid can control the strength of the solvent in a precisely controlled manner. As opposed to conventional solvent extraction, any residual CO2 left in the extract after separation is inert and non-toxic, so that the human consumption of the material is not harmful. The extraction process illustrated in Figure 2 is typically performed in a very high-pressure, thick-walled, rounded chamber designed to withstand pressures of up to about 120 MPa (1450-17.400 psi), more particularly up to about 70 MPa ( 10,150 psi). The chamber has openings to add a suitable load of green vegetable protein concentrate in the upper part and for the removal of the load after extraction in the lower part. Appropriate pump and pipe systems direct the supercritical fluid carbon dioxide at the bottom of the chamber so that the liquid will flow through the bed of green plant material and to the top of the chamber for release to a collection device. During or after the release of the extract to the collection device, the supercritical fluid can be depressurized to below the desired pressure to collect the desired extract. In one embodiment, the extraction method is carried out against the flow of the SCF in relation to the movement of the green plant material. Importantly, temperature and pressure can be controlled with conventional devices such as pumps, valves and / or conventional heat exchangers before, during and / or after extraction to optimize the concentration, combination or mixture of lutein or other nutrients in a particular extraction. After leaving the extraction chamber, a pressure reducing valve can be placed before collection by the collection device to effect the release or precipitation of the desired extract alone, or on a specific carrier material in the collection device. A double valve at the bottom of the collection device allows periodic removal of the extract (with or without the carrier). The liquid carbon dioxide vented from the top of the collection device at a reduced pressure can be recycled to a filter system and be recompressed at high pressure for use in a second extraction function in the extraction vessel. The extraction continues until an appropriate degree of dry product is isolated from the green plant material that is processed. The volume of SCF needed for the desired extraction depends on the pressure and the temperature used for each product obtained. Typically 5 - 50 cubic feet of SCF are needed for each cubic foot of extracted vegetable concentrate. The ratio between the supercritical fluid volume and the green plant material can be mentioned as the ratio of solvent to feed, and can more particularly vary from 10: 1 to 50: 1. In one embodiment, the supercritical extraction is carried out under at least two different pressure and temperature conditions in the extraction chamber. At a pressure and temperature, a first extract containing substantial amounts of carotene can be obtained. At a second pressure and temperature, a second extract containing substantial amounts of lutein can be obtained. In one embodiment the second extract may be substantially free of ß-carotene. For example, the second extract may have less than 10 percent ß-carotene, more particularly less than 5 percent. This can be achieved by effecting the first extraction until the green plant material is substantially free of a-carotene, and then subsequently making the second extraction until the extraction of the desired lutein is completed. In one example, the first extraction may be carried out at a pressure of between about 10 and about 40 MPa, more particularly between about 15 and about 35 MPa. The second extraction can be carried out at a pressure between about 41 and about 80 MPa, more particularly between about 55 and about 80 MPa. Both extractions can be carried out at between approximately 31 ° C and approximately 100 ° C. The extractions can be carried out at the same or at different temperatures. For example, the first extraction temperature may vary from about 31 ° C to about 40 ° C. The second extraction temperature may vary from about 65 ° C to about 75 ° C depending, at least in part, on the extraction pressure, the green plant material, the supercritical fluid volume and / or a co-solvent of drag. As is evident from the foregoing, the second extraction by supercritical fluid (or other extractions) does not necessarily have to be carried out both at a different temperature and at a different pressure than those of the first extraction. Rather, one or both of the temperature and pressure can be changed between extractions to achieve the desired result. Accordingly, as used herein, the changes to "pressure and temperature," or "pressure and temperature conditions," refer to changes in the general condition under which the extraction is effected, preferably to changes in both the temperature as in the pressure. In an alternative embodiment, multiple extractions at multiple pressures and temperatures can be performed to obtain extracts containing concentrations of a desired nutrient or nutrients that are different from the nutrient or nutrient concentrations that can be obtained by merely performing a simple extraction. For example, a first extraction can be carried out at a first pressure to obtain a first extract. A second extraction can then be carried out at a second pressure and temperature to obtain a second extract. The first extract may contain a substantial concentration of β-carotene, while the second extract may contain a higher concentration of lutein than the first extract, while also optionally including a controlled amount of β-carotene and / or fatty acid. Multiple similar extraction methods can be used to achieve a desired concentration (or range of concentrations) of a nutrient mixture in a particular extract that could not be obtained using a single extract of the green plant material. In this way, an extraction can be obtained by having a controlled combination of lutein, β-carotene and / or fatty acids. This may be beneficial for certain applications, because it has been recognized that β-carotene and lutein are important in preserving ocular health because lutein is concentrated in the macula and β-carotene is converted to Vitamin A, which It is critical for night vision and general retina health. In addition, fatty acids can improve the appearance of lutein and ß-carotene. Therefore, a combined mixture of β-carotene with an adequate concentration of fatty acids is a good nutritional supplement to maintain and / or improve ocular health. In one embodiment, the desired extract may include high concentrations of lutein, with only trace amounts of β-carotene and fatty acids. In another embodiment, the desired extract may include a controlled concentration of β-carotene, lutein and fatty acids. For example, the extract may include between about 10 and 90 weight percent, more particularly about 40 and about 60 weight percent lutein, between about 10 and 90 weight percent, more particularly about 40 and about 60 percent in weight of β-carotene, and between approximately 5 and 20 weight percent of fatty acids. Additionally, the supercritical extraction process of embodiments of the present invention can be used to eliminate other undesirable materials, including chlorophyll, aromas and odor-producing compounds, and hormones such as cumesterol. Accordingly, in one embodiment, at least one extract includes lutein, but is substantially free of hormones such as cumesterol, odor and aroma producing compounds and / or chlorophyll. Although the pressure, temperature and volume at which the supercritical extractions are made are related, each of these variables or conditions can be adjusted and / or optimized independently to produce one or more extracts that have specific concentrations of nutrients and / or other desired substituents. As an example, if a total separation of β-carotene from alfalfa is desired, an extraction by initial supercritical fluid can be carried out under low temperature and / or low pressure (32 ° C); 20 MPa; 20-50 volumes of CO2), so that substantial portions of β-carotene will be isolated and concentrated in the extract. If higher temperatures and / or higher pressures are used (43 ° C, 50 MPa, 20-30 volumes), lutein and ß-carotene can be concentrated in a single extract. In addition, the volume of supercritical fluid needed to extract the desired nutrients may depend on the pressure and the temperature at which the extraction is effected. For example, under conditions of low temperature and pressure, it may be desirable to use a larger volume of supercritical fluid to obtain the desired extract. However, under higher temperature and pressure conditions, a lower volume of supercritical fluid may be required to obtain a desired extract. In this way, it is possible to adjust or optimize the pressure, temperature and / or extraction volume to obtain extractions having the desired type, concentration and / or purity of nutrients.

In certain embodiments, it may be desirable to make at least a third extract at a third temperature or pressure. For example, saponins can be isolated and extracted under conditions of pressure and / or temperature higher than lutein and ß-carotene.

Post-Extraction Processing Optionally, after separation, the extract may be further processed to produce a desired final product. For example, a secondary column fractionation step can be used to further concentrate and purify lutein or β-carotene. Additionally, the first and second extracts can be purified with simple non-toxic solvents such as food-grade ethanol, vegetable oil, or water to provide a substance that is crystalline and essentially pure and free of any potentially toxic chemical, yet at a trace level. Typically, lutein is concentrated at 5-50% concentration in oils or in dry form for bulk marketing. In one embodiment, the first and second extracts are combined before or after separation in order to provide a final product having a controlled concentration of carotene and lutein. Advantageously, in embodiments using multiple extractions to obtain a controlled concentration of carotene and lutein (or other nutrients), this post-extraction processing can be minimized or completely eliminated.

Lutein and / or ß-carotene can be further processed by combination or milling with a suitable base material (eg, green vegetable protein concentrate or other combined agents) to form a final product for human consumption. This combination or milling step can take place in the collection device where the extract is precipitated in the base material. The double valve at the bottom of the collection device can be activated to release the combined extract. In this way, protein concentrates or mixing agents can be used as an anchoring agent in the collection containers at lower pressure.

Final Products The extract can be combined with a suitable carrier to form a final product. The final product can be a powder, a bound powder or an edible oil solution that includes the extract. A protein matrix or pearls can be produced to protect the extract from deterioration or oxidation. It can be analyzed for specific carotenoid content and then mixed with alfalfa or natural fillers based on vegetables, sugars, gelatins, or starches to form a desired standardized dry product. In one embodiment, the extract is combined with a fraction rich in green chloroplasts of alfalfa (which can be used as the initial green plant material) so that the final product will contain only an ingredient of unique origin and can be marked as based on 100% alfalfa. The use of the green chloroplast fraction of alfalfa as the carrier in the final product is nutritionally beneficial because of the high content of proteins, vitamins, amino acids, chlorophyll and other compounds useful in the fraction in addition to the presence of concentrated lutein. In addition, the final product is then derived from a vegetable product of unique origin, without additional fillers or additives. The invention is further described in the following Example.

EXAMPLE Alfalfa was recently crushed through a hammer mill to break plant cells. The end speed of the hammers was adjusted to 15,000 feet per minute to crush the wet green material (80% moisture) without causing the material to be punctured or broken into smaller pieces. The crushed material was run through a single screw press (6") (Model Number VP6, available from Vincent Corp., Tampa, FL) so that the exit restriction (adjusted to 25 psi) produces high continuous pressure for Perform separation of green vegetable juices from vegetable fibers The wide drum spindle has a fine drum spindle to allow the juice to flow out of the fiber.The ratio of juice to fiber was about 1: 1, however, the yield of juice to fiber will be less if the initial material is old or more mature, or if it is naturally drier than fresh cultivated pre-flowering alfalfa. The juice was adjusted to a pH of 8.0 with ammonia water, and immediately heated from room temperature with a double evaporator system with a propane burner so that the juice is heated in 5-10 minutes after production between 82 - 85 ° C to cause thermal coagulation of the green and white proteins (cytoplasmic) The green protein clot was separated with a screw-type weir to separate the green "rennet" from brown waste vegetable juices. 'rennet' of wet green protein (ie the green plant material) was immediately dried in a continuously drilled controlled temperature zone dryer, so that limited heat (below 85 ° C) with air limited to 5-10% of Relative humidity produced a dry granular material.The moist protein rennet started at approximately 75% humidity and dried up to 8% moisture.This material was then extracted or stored in bags or containers that xcluyen oxygen in the dark at room temperature until it was extracted. The green plant material was then transferred to a very high pressure extraction chamber (approximately 5 cm x 50 cm) having thick rounded walls, and which is designed to withstand pressures of up to 70 MPa (10,150 psi). The chamber was brought to pressure and temperature with carbon dioxide fluid at 20 MPa at 30 ° C. The temperature and pressure of the SCF stream with a liquid ethanol entrainer at 3% by volume injected were regulated by means of a high pressure carbon dioxide pump and water-controlled heat exchanger in shell and tube system. This extraction continued until the beta-carotene was removed (approximately 27 bed volumes) as measured in the sampling of the lateral port at the top of the column exit line. The pressure was then increased to 65 MPa to extract the lutein from the green plant material with approximately 20 bed volumes. The desired compounds were collected after extraction in a small but high (1 meter) tower with reduced pressure through reducing valves so that the fractions of beta-carotene and lutein were collected in chambers with dry green protein powder at 10 MPa and 40 MPa respectively. The lower quarter of the collection vessel has large valves to allow the desired fractions to exit into the protein fractions so that after separation of the fractions, the lower chamber of collection was released from the seal and the pressure was released to remove the final products. The yield in this example was 2.6 grams of beta carotene and 2.4 grams of lutein per kilogram of initial dry material (6% moisture). The products were tested for purity without mixing with the green protein fraction with HPLC columns of silica gel and were 80% and 72% pure carotene and lutein respectively. Drying green protein is critical in the preservation of dry end products since dry materials ranged from 0.6 to 3.4 grams of each carotenoid that was measured with high performance liquid chromatography (HPLC) with known purity standards (available from Sigma Chemical, St.

Louis, MO).

Claims (40)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for obtaining lutein from green plant materials comprising: making a first extraction by supercritical fluid of a green plant material at a first pressure and temperature to obtain a first extract; perform a second supercritical fluid extraction of the green plant material at a second pressure and temperature to obtain a second extract that includes a higher concentration of lutein than the concentration of lutein in the first extract, and separate the second extract from the supercritical fluid.

2. The method according to claim 1, further characterized in that the green plant material is derived from green vegetables or fractions of dried green vegetables.

3. The method according to claim 1, further characterized in that the green plant material comprises a fraction of alfalfa rich in dry or wet chloroplasts.

4. The method according to claim 1, further characterized in that the first extract comprises β-carotene, at least one fatty acid, or both.

5. The method according to claim 1, further characterized in that the first extraction by supercritical fluid reduces the concentration of β-carotene, fatty acids, or both in the green plant material.

6. The method according to claim 1, further characterized in that the first extraction by supercritical fluid reduces the concentration of cumesterol in the green plant material.

7. The method according to claim 1, further characterized in that the first extraction by supercritical fluid reduces the concentration of chlorophyll in the green plant material.

8. The method according to claim 1, further characterized in that the first extraction by supercritical fluid reduces the concentration of the odor or compounds that produce aromas in the green plant material.

9. The method according to claim 1, further characterized in that the second extract is substantially free of carotene.

10. The method according to claim 1, further characterized in that the first extract comprises β-carotene.

11. The method according to claim 1, further characterized in that the first extract comprises between about 10 and about 60 weight percent of β-carotene.

12. The method according to claim 1, further characterized in that the concentration of β-carotene in the first extract is lower than in the second extract.

13. The method according to claim 1, further characterized in that the second extract comprises β-carotene.

14. The method according to claim 1, further characterized in that the second extract comprises between 10 and about 60% by weight of β-carotene.

15. The method according to claim 1, further characterized in that the concentration of β-carotene in the second extract is lower than in the first extract.

16. The method according to claim 1, further characterized in that the second extract comprises at least one fatty acid.

17. The method according to claim 16, further characterized in that the second extract comprises less than about 20 weight percent fatty acids.

18. The method according to claim 1, further characterized in that the second extract is free of hormones.

19. The method according to claim 1, further characterized in that the second extract is free of cumesterol.

20. The method according to claim 1, further characterized in that the second extract is substantially free of chlorophyll.

21. The method according to claim 1, further characterized in that the first and second pressures are between about 10 and about 120 MPa.

22. The method according to claim 1, further characterized in that the first pressure is lower than the second pressure.

23. The method according to claim 1, further characterized in that the first pressure is between about 10 and about 40 MPa.

24. The method according to claim 1, further characterized in that the second pressure is between about 41 and about 80 MPA.

25. The method according to claim 1, further characterized in that the first pressure is between about 15 and about 35 MPa and the second pressure is about 55 and about 80 MPa.

26. The method according to claim 1, further characterized in that the first and second extraction by supercritical fluid takes place at between approximately 31 ° C and approximately 100 ° C.

27. The method according to claim 1, further characterized in that the first temperature is lower than the second temperature.

28. The method according to claim 1, further characterized in that the first temperature is between about 31 ° C and about 40 ° C, and the second temperature is between about 65 ° C and about 75 ° C.

29. The method according to claim 1, further characterized in that the first and second extraction by supercritical fluid take place at a substantially constant temperature.

30. The method according to claim 1, further characterized in that the first and second extraction by supercritical fluid are effected with a supercritical fluid and a trawl.

31. The method according to claim 1, further characterized in that the second extract from supercritical fluid comprises subjecting the second extract to a postextraction pressure that is lower than the second pressure.

32. The method according to claim 31, further characterized in that the post-extraction pressure is between about 15 and about 45 MPa.

33.- The method according to claim 1, further characterized in that it comprises separating the first extract from the supercritical fluid.

34.- The method according to claim 33, further characterized in that it comprises separating the first extract from the supercritical fluid by subjecting the first extract to a post-extraction pressure that is lower than the first pressure.

35. - The method according to claim 33, further characterized in that it comprises combining the first and the second extract.

36. The method according to claim 1, further characterized in that it further comprises processing the first or the second extract by fractionation on a column of silica gel.

37. The method according to claim 1, further characterized in that it additionally comprises processing the first or the second extract by purification with a non-toxic solvent.

38. The method according to claim 1, further characterized by additionally comprising processing the first or second extract by mixing with the dried green protein. 39.- The method according to claim 1, further characterized in that it comprises performing at least a third extraction by supercritical fluid of the green plant material at a third pressure and temperature to obtain at least a third extract. The method according to claim 39, further characterized in that the third pressure is lower or higher than the first and second pressures. 41. The method according to claim 39, further characterized in that the third pressure is higher than the first and second pressures. 42. The method according to claim 39, further characterized in that the third extract comprises at least one saponin. 43.- A method for obtaining a plurality of extracts from green plant material, comprising: making a first extraction by supercritical fluid of green plant material at a first pressure and temperature to obtain a first extract; performing at least one additional supercritical fluid extraction of the green plant material at at least one additional pressure and temperature to obtain at least one additional extract, wherein at least one of the additional extracts includes a higher concentration of lutein than in the first extract; and separating at least one of the additional extracts from the supercritical fluid. 44. The method according to claim 43, further characterized in that at least one of the extracts includes lutein, but is substantially free of carotene. 45. The method according to claim 43, further characterized in that at least one extract comprises β-carotene. 46. The method according to claim 43, further characterized in that at least one extract comprises alpha-carotene. 47. The method according to claim 43, further characterized in that at least one extract comprises at least one fatty acid found in the green plant material. 48. The method according to claim 47, further characterized in that the green plant material is derived from alfalfa, and the fatty acid comprises linolenic acid, linoleic acid, palmitic acid, oleic acid or combinations thereof. 49. The method according to claim 43, further characterized in that at least one extract comprises antoxanthin, zeaxanthin, astaxanthin, canthaxanthin, capsorubin, cryptoxanthin or combinations thereof. 50.- The method according to claim 43, further characterized in that at least one of the extracts comprises a hormone. 51. The method according to claim 50, further characterized in that the hormone comprises cumesterol. 52. The method according to claim 43, further characterized in that at least one extract comprises a mixture of at least two nutrients. 53. The method according to claim 43, further characterized in that at least one of the additional extracts comprises a mixture of lutein and β-carotene. 54. The method according to claim 53, further characterized in that the additional extract comprises between about 20 and about 60% by weight of lutein and between about 20 and about 60% by weight of β-carotene. 55.- The method according to claim 53, further characterized in that the additional extract additionally comprises at least one fatty acid. 56. The method according to claim 55, further characterized in that it comprises between about 5 and about 20 weight percent of at least one fatty acid. 57. A method for isolating lutein from green plant materials comprising: making a first supercritical fluid extraction of a green plant material at a first pressure to obtain a first extract; performing a second supercritical fluid extraction of the green plant material at a second pressure to obtain a second extract that includes lutein, but is substantially free of carotene; and separating the second extract from the supercritical fluid. 58. The method according to claim 57, further characterized in that it comprises separating the first extract from the supercritical fluid.