Carotenoids crystals containing lutein with high amounts of trans-zeaxanthin and a process of isolation thereof

Abstract

The present invention relates to carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3'R) wherein the lutein and trans-zeaxanthin (3R, 3'R) are in a ratio in the range of 9:1 to 12:1 and to a process of isolation thereof More particularly, the invention relates to carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3'R) derived from non-mutant marigold flowers.

Description

[0001] CAROTENOIDS CRYSTALS CONTAINING LUTEIN WITH HIGH AMOUNTS OF TRANS-ZEAXANTHIN AND A PROCESS OF ISOLATION THEREOF FIELD OF INVENTION

[0002]

[0001] The present invention relates to carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) wherein the lutein and trans-zeaxanthin (3R, 3 ’R) are in a ratio in the range of 9: 1 to 12: 1. More particularly, the invention relates to carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) derived from non-mutant marigold flowers and to a process of isolation thereof.

[0003] BACKGROUND OF THE INVENTION

[0004]

[0002] Carotenoids are a class of natural fat-soluble pigments found principally in plants, algae, and photosynthetic bacteria, where they play a critical role in the photosynthetic process. They are also produced by several non-photosynthetic bacteria, yeasts, and molds. Although animals appear to be incapable of synthesizing carotenoids, many animals incorporate carotenoids from their diet. Carotenoids are categorized into two classes xanthophylls and carotenes being defined by their chemical structure. The hydrocarbon carotenoids are known as carotenes, while oxygenated derivatives of these hydrocarbons are known as Xanthophylls. Beta-carotene, the principal carotenoid in carrots, is a familiar carotene, while Lutein, the major yellow pigment of marigold petals, is a common xanthophyll.

[0005]

[0003] The major xanthophylls are lutein, zeaxanthin, Beta-cryptoxanthin, astaxanthin, of which lutein, zeaxanthin, beta-cryptoxanthin are classified as primary xanthophylls as they are produced directly, whereas astaxanthin and canthaxanthin are classified as secondary xanthophylls as they are synthesized from primary xanthophylls. Thexanthophylls found in the bodies of animals, and in dietary animal products, are ultimately derived from plant sources in the diet. For example, the yellow color of chicken egg yolks, fat, and skin comes from ingested xanthophylls (primarily lutein, which is often added to chicken feed for this purpose). Within animals, carotenoids provide bright coloration, serve as antioxidants, and can be a source for vitamin A activity (Ong and Tee 1992; Britton et al. 1995).

[0006]

[0004] Lutein is found in most fruits and vegetables, but green and yellow foods have the highest amounts. One of the main natural sources of carotenoids is the marigold (Tagetes erecta) flower, which has been extensively used by industry for the production of food colorants or supplements. Zeaxanthin is a naturally occurring pigment present in egg yolk, in maize, marigold (Tagetes erecta) flowers and in fruits such as oranges, peaches, especially in Lycium fruits (Lycium barbarum, Lycium Chinese). While lutein is relatively abundant in the food we eat, zeaxanthin is not that easily obtained through a well-balanced diet and thus should be added as a food additive. The yellow color of the human macula lutea in the retina of the eye comes from the lutein and zeaxanthin it contains, both xanthophylls again requiring an exogenous source in the human diet to be present in the eye.

[0007]

[0005] Lutein and zeaxanthin supplementation is known to have various beneficial effects on the brain, eyes as well as skin. These carotenoids act as anti-inflammatories as well as antioxidants. There are numerous studies that have assessed how lutein and zeaxanthin supplementation affect the vision of healthy individuals. Low macular levels of lutein and zeaxanthin have been shown to be correlated to the development of age-related macular degeneration. Further, a number of studies have shown that supplementation with lutein and zeaxanthin when given in a ratio of 10:1 to 10:2, improve glare sensitivity and other aspects of vision in healthy adults. A study byMadhavan el al., has evaluated the modulatory effect of oil-soluble dietary carotenoid supplement constituting lutein and zeaxanthin in the ratio of 10:1 on anti-oxidant enzymes and macular pigments in the serum and macula of the Swiss albino rats and the study provides a strong claim for the anti-oxidant effect of the oil-soluble dietary carotenoid supplement, and thus substantiates its use in the prevention of phototoxic damage to the eye on long-term supplementation.

[0008]

[0006] Also, several studies have shown that central nervous system levels of lutein and zeaxanthin are related to brain function. Lutein and zeaxanthin are also important for the health of the skin. They help protect against the damaging effects of another portion of the electromagnetic spectrum: ultraviolet light. Animal studies have shown that supplementation of lutein and zeaxanthin reduces inflammation, edema, reactive oxygen species production and epidermal hyperproliferation with exposure to UV-B radiation.

[0009]

[0007] Although lutein and zeaxanthin may be obtained from certain fruits and vegetables, the isolation of lutein from extracts of marigold flowers is most common. The carotenoid crystals isolated from marigold flowers contain approximately 80% lutein, including its derivatives, and 5% Zeaxanthin. A number of methods have been used for isolation of carotenoids crystals from marigold flowers.

[0010]

[0008] U.S. Patent No. 5,648,564 to Kemin Industries Inc. discloses a process for saponification of the marigold oleoresin with an aqueous alkali diluted with propylene glycol, resulting in the formation of lutein crystals.

[0011]

[0009] U.S. Patent No. 6,262,284 to University of Maryland discloses a process for extracting, saponifying, and isolating lutein and zeaxanthin, and a mixture of severalrare carotenoids in high purity from plants such as Marigold flowers, Lycium Chinese Mill, and green plants.

[0012]

[0010] U.S. Patent No. 6,380,442 to BioActives, LLC, discloses a process for isolating and purifying mixed carotenoids containing higher concentrations of specific compounds, such as all-trans lutein, from marigold oleoresin through hydrolysis by using iso-propyl alcohol with saponification. Further, the process involves cooling the reaction mixture to room temperature. The final product contains between about 53% and 95% (weight / weight) total carotenoids and between about 50% and 88% all-trans lutein (based on spectrophotometry and reverse phase HPLC).

[0013] [Oil] Commercially available xanthophyll pigments are extracted from a marigold hybrid (Hybrid 50011, U.S. Pat. No. 6,894,208) which typically has atotal xanthophyll composition of 94% lutein (L) and 6% zeaxanthin (Z); with an average L:Z ratio range of 15:1.

[0014]

[0012] US Patent No. 7,173,145 to Khachik discloses a process for extracting and isolating carotenoid esters or carotenoids wherein the zeaxanthin esters were isolated and purified from the berries of Lycium Chinese Mill (LCM berries).

[0015]

[0013] US Patent No. 6,784,351 to Ball Horticultural Co. discloses marigold plants whose petals, leaves or both flower petals and leaves contain one or more of an enhanced zeaxanthin ratio, an enhanced neoxanthin plus violaxanthin ratio, an enhanced P-carotene ratio, an enhanced a-cryptoxanthin ratio an enhanced phytoene ratio or an enhanced phytofluene ratio compared to such a ratio present in a non-mutant marigold. In addition, the flower petals typically contain a measurable amount of zetacarotene ((^-carotene), whereas that compound is not measurable; i.e., is present at less than 0.1 percent, in the petals of a non-mutant marigold plant.

[0014] Further, zeaxanthin may also be obtained from sources other than plants. For example, US Publication No. 20070190595A1 discloses a process of obtaining zeaxanthin in a high yield from blue-green algae. Zeaxanthin and zeaxanthin-enriched products obtained by the process, which are suitable for use as dietary supplements and / or food additives, are also described. Further, US Patent No. 6,376,722 discloses a lutein to zeaxanthin isomerization process which includes a step of saponification or alkali treatment of a plant extract which contains lutein, followed by a step of heating under controlled conditions in the presence of a surfactant having a hydrophilic-lipophilic balance from 1 to 40 as a catalyst for the lutein to zeaxanthin isomerization reaction at a temperature from 70°C to 140°C in order to obtain a reaction product with high zeaxanthin concentrations of up to 80% with respect to total carotenoids. As the process disclosed includes isomerisation at higher temperatures and usage of alkali, the carotenoids crystals obtained contain zeaxanthin in the form of meso-Zeaxanthin which is 3R, 3’S.

[0016]

[0015] US Patent No. US10,485,833 to OmniActive Health Technologies Inc. discloses a composition comprising of trans-lutein, (R,R)-zeaxanthin and (R,S)-zeaxanthin wherein the process for preparing the composition involves isomerization of lutein to zeaxanthin isomers.

[0017]

[0016] More recently, attempts have been made to produce a new and distinct inbred plant of Tagetes erecta named KI4662 and characterized by elevated levels of zeaxanthin and little or no lutein as is disclosed in US Patent No. US 9,848,5462 to Kemin Industries Inc. and the marigold flowers would therefore be the mutant variety.

[0018]

[0017] As is evident from the above discussion, it is clear that in order to obtain crystals with high amount of trans-zeaxanthin, either the raw material used is berries of Lycium Chinese Mill (LCM berries) or a mutant variety of marigold is utilized. Further, for aneconomic production of zeaxanthin substitutes, methods such as isomerisation of marigold lutein to zeaxanthin is carried out, wherein the isomerisation process produces meso-zeaxanthin (3R, 3'S) instead of trans-Zeaxanthin (3R, 3'R) isomeric form of zeaxanthin that is found in nature.

[0019]

[0018] However, there remains a need for obtainment of carotenoids crystals from naturally occurring non-mutant marigold flowers such that the carotenoids crystals contain lutein with high amounts of trans-zeaxanthin (3R,3’R) in it its natural form and an improved process of isolation thereof.

[0020]

[0019] Therefore, the objective of the present invention is to provide carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) wherein the lutein and trans-zeaxanthin (3R, 3’R) are in a ratio in the range of 9:1 to 12:1. Another objective of the present invention is to provide a process of isolation of carotenoids crystals from naturally occurring non-mutant plant source, preferably marigold, wherein the resultant carotenoids crystals contain lutein and a high amount of trans-zeaxanthin (3R,3’R).

[0021] SUMMARY OF THE INVENTION

[0022]

[0020] In an embodiment of the invention, there are provided carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) wherein the lutein and trans-zeaxanthin (3R, 3’R) are in a ratio in the range of 9: 1 to 12:1.

[0023]

[0021] According to an embodiment of the invention there are provided carotenoids crystals obtained from non-mutant plant oleoresin.

[0024]

[0022] In an embodiment of the invention the carotenoids crystals are isolated from a plant oleoresin such as marigold oleoresin, preferably from oleoresin derived from non-mutant marigold flowers.

[0023] According to an embodiment of the invention a process for isolation of carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R,3’R) is provided.

[0025]

[0024] In an embodiment of the invention the process of isolation of carotenoids crystals comprises of taking a plant oleoresin as the raw material. Preferably, from oleoresin derived from non-mutant marigold flowers.

[0026]

[0025] In an embodiment of the invention the process of isolation of carotenoids crystals comprises of taking a pre-defined amount of a plant oleoresin, homogenizing it, and subjecting it to a saponification process using an alcoholic alkali. The saponified reactant mass is then subjected to crystallization by adding a suitable solvent followed by addition of ethyl alcohol / ethanol which is followed by filtering to recover the crystals. The carotenoids crystals are neutralised using hot water and finally, the wet crystals are collected and dried to obtain dried carotenoids crystals. The carotenoids crystals obtained contain Lutein and high amounts of trans-Zeaxanthin (3R,3’R), preferably the ratio of lutein to trans-zeaxanthin is in the range of 9: 1 to 12: 1.

[0027]

[0026] In an embodiment of the invention the solvent for crystallization of the reactant mass may be selected from the group consisting of isopropyl alcohol, ethyl alcohol, butyl alcohol, iso-amyl alcohol, 1 -propanol, iso propyl acetate, ethyl acetate, butyl acetate, iso butyl acetate, methyl acetate, ethyl acetate, iso-butanol, heptane, pentane and other suitable al cohol / sol vents.

[0028]

[0027] In another embodiment of the invention the process of isolation of carotenoids crystals comprises of taking a pre-defined amount of a plant oleoresin, homogenizing it, and subjecting it to a saponification process using an alcoholic alkali. The saponified reactant mass is then subjected to crystallization by adding demineralized hot water.The diluted mass with carotenoids crystals is filtered to recover the crystals. The carotenoids crystals are then mixed with an alcohol wherein the alcohol could be selected from ethyl alcohol and / or iso-propyl alcohol. The mass is then filtered and wet crystals collected are mixed with a suitable solvent followed by addition of ethanol which is followed by filtering to recover the crystals and finally, the wet crystals are collected and dried to obtain dried carotenoids crystals. The carotenoids crystals obtained contain Lutein and high amounts of trans-Zeaxanthin (3R,3’R), preferably the ratio of lutein to trans-zeaxanthin is in the range of 9: 1 to 12: 1.

[0029]

[0028] In an embodiment of the invention the solvent for crystallization of the reactant mass may be selected from the group consisting of isopropyl alcohol, ethyl alcohol, butyl alcohol, iso-amyl alcohol, 1 -propanol, iso propyl acetate, ethyl acetate, butyl acetate, iso butyl acetate, methyl acetate, ethyl acetate, iso-butanol, heptane, pentane and other suitable al cohol / sol vents.

[0030]

[0029] According to an embodiment of the invention, the alcoholic alkali used in the saponification process could be selected from ethanolic potassium hydroxide and ethanolic sodium hydroxide.

[0031]

[0030] In an embodiment of the invention, the resulting carotenoid crystals contain a minimum of 85% carotenoids determined by spectrophotometer of which about 90.0% is all trans-Lutein and about 7.0% to 9.5% all trans-Zeaxanthin along with minute quantities of Beta-carotene and Cryptoxanthin determined by HPLC. Further, the chemical recovery of the final product is in the range of 45 to 70.0% while the physical recovery of the final product is approximately 7 to 12%.

[0032]

[0031] In another embodiment of the invention, the carotenoid crystals have a purity of at least 85%.

[0032] According to yet another embodiment of the invention, the carotenoids crystals containing high amounts of trans-zeaxanthin are formulated in the form of powder, beadlets, granules, oil dispersion or water dispersion or any other suitable form.

[0033]

[0033] According to another embodiment of the invention, the carotenoids crystals containing high amounts of trans-zeaxanthin (3R,3’R) are used in food and beverages, dietary supplements, nutraceutical or cosmeceutical applications.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035]

[0034] Discussed below are some representative embodiments of the present invention. The invention in its broader aspects is not limited to the specific details and representative methods. The illustrative examples are described in this section in connection with the embodiments and methods provided. The invention according to its various aspects is particularly pointed out and distinctly claimed in the attached claims read in view of this specification. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present application. The present application is not intended to be limited to the embodiments shown but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0036]

[0035] References to “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” “some embodiments,” “embodiments of the invention,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every possible embodiment of the invention necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplaryembodiment,” “an embodiment,” do not necessarily refer to the same embodiment, although they may. Moreover, any use of phrases like “embodiments” in connection with “the invention” are never meant to characterize that all embodiments of the invention must include the particular feature, structure, or characteristic, and should instead be understood to mean “at least some embodiments of the invention” includes the stated particular feature, structure, or characteristic.

[0037]

[0036] Similarly, the words "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. Also, references to the term “comprising” may also include the case of “containing”, “consisting of’ and “consisting essentially of’ and may be used interchangeably. Likewise, the terms “include”, “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. However, the embodiments provided by the present disclosure may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment defined using the term "comprising" is also a disclosure of embodiments “consisting essentially of’ and “consisting of’ the disclosed components. Where used herein, the term “example”, particularly when followed by a listing of terms, is merely exemplary and illustrative, and should not be deemed to be exclusive or comprehensive. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein unless explicitly indicated otherwise.

[0038]

[0037] Unless otherwise defined, each technical or scientific term used herein has the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used in the description and claims, the singular form “a”, “an” and “the” include both singular and plural references unless the context clearly dictates otherwise. For example, the term “sensor” may include, and is contemplated to include a plurality of sensors. At times, the claims and disclosure may include terms such as “aplurality”, “one or more,” or “at least one; however, the absence of such terms is not intended to mean, and should not be interpreted to mean, that a plurality is not conceived.

[0039]

[0038] The terms trans-Lutein, lutein, t-lutein are interchangeably used herein to refer to the xanthophyll ‘Lutein’. The terms trans-Zeaxanthin, t-zeaxanthin, are interchangeably used herein to refer to the isomeric form 3R, 3’R of the xanthophyll ‘Zeaxanthin’ .

[0040]

[0039] The present invention provides carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) wherein the lutein and trans-zeaxanthin (3R, 3’R) are in a ratio in the range of 9:1 to 12:1. The present invention also provides a novel process of isolation of carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) derived from non-mutant marigold flowers.

[0041]

[0040] According to an embodiment of the invention there are provided carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R, 3’R) wherein the lutein and trans-zeaxanthin (3R, 3’R) are in a ratio in the range of 9:1 to 12:1 in the obtained carotenoids crystals.

[0042]

[0041] According to an embodiment of the invention there are provided carotenoids crystals obtained from non-mutant marigold flowers.

[0043]

[0042] According to an embodiment of the invention a process for isolation of carotenoids crystals containing lutein with high amounts of trans-zeaxanthin (3R,3’R) is provided.

[0044]

[0043] According to an embodiment of the invention the process of isolation of carotenoids crystals comprises of taking a plant oleoresin as the raw material.

[0044] In an embodiment of the invention the plant oleoresin is derived from marigold flowers.

[0045]

[0045] According to an embodiment of the invention the plant oleoresin is derived from non-mutant marigold flowers.

[0046]

[0046] According to an embodiment of the invention the carotenoids crystals obtained contain Lutein and trans-Zeaxanthin (3R,3 ’R) in a ratio in the range of 9: 1 to 12:1.

[0047]

[0047] The process of isolation of carotenoids crystals comprises of the following steps:

[0048] (a) taking a pre-defined amount of a plant oleoresin in a reactor with an agitator and homogenizing the oleoresin for 10 minutes at a temperature of 40°C under stirring;

[0049] (b) adding an alcoholic alkali into the reaction vessel slowly, containing the homogenized mass of oleoresin, at a temperature of 75°C for 30 minutes to saponify the mass and ensuring the degree of saponification to be more than 99% by HPLC,

[0050] (c) subjecting the reactant mass to crystallization by adding a suitable solvent under continuous stirring at a temperature of 60°C - 75°C for 30-60 minutes followed by adding ethanol / ethyl alcohol under continuous stirring at a temperature of 60°C - 75°C for 30-40 minutes; (d) filtering the diluted mass in a filter to recover the crystals and adding hot water at a temperature in the range of 60°C to 70°C, to wash away the unwanted impurities and to bring down the pH of the effluent to neutral around 7.0;

[0051] (e) collecting the wet crystals from the filter and drying in a suitable dryer at a temperature of around 55°C to 60°C for 3-5 hours to obtain driedcarotenoids crystals. The carotenoids crystals obtained contain Lutein and high amounts of trans-Zeaxanthin (3R,3’R), preferably the ratio of lutein to t-zeaxanthin is in the range of 9: 1 to 12:1.

[0052]

[0048] In an embodiment of the invention the solvent for crystallization of the reactant mass may be selected from the group consisting of isopropyl alcohol, ethyl alcohol, butyl alcohol, iso-amyl alcohol, 1 -propanol, iso propyl acetate, ethyl acetate, butyl acetate, iso butyl acetate, methyl acetate, ethyl acetate, iso-butanol, heptane, pentane and other suitable al cohol / sol vents.

[0053]

[0049] According to another embodiment of the invention the process of isolation of carotenoids crystals may comprise of the following steps:

[0054] a. taking a pre-defined amount of a plant oleoresin in a reactor with an agitator and homogenizing for 10 minutes at a temperature of 40°C under stirring;

[0055] b. adding an alcoholic alkali into the reaction vessel slowly, containing the homogenized mass of oleoresin, at a temperature of 75°C for 30 minutes to saponify the mass and ensuring the degree of saponification to be more than 99% by HPLC;

[0056] c. subj ecting the mass to crystallization by adding demineralized hot water maintained at a temperature of 60°C - 75°C to the reacted mass while stirring for 30 minutes;

[0057] d. filtering the diluted mass with carotenoid crystals in a filter to recover the crystals and neutralizing the carotenoids crystals using hot water at a temperature of 60°C to 70°C, to wash away the unwanted impurities and bring down the pH of the effluent to neutral around 7.0;e. filtering the wet crystals, collecting them from the filter and mixing an alcohol at a temperature of 65°C for 30 minutes. The alcohol could be selected from ethyl alcohol and / or iso-propyl alcohol;

[0058] f. filtering the mass under vacuum at a temperature of 65°C, collecting the wet crystals and mixing with a solvent maintained at a temperature of 75°C for 30 minutes;

[0059] g. filtering the mass under vacuum and collecting the wet crystals and adding ethyl alcohol maintained at a temperature of 60°C for 30 minutes;

[0060] h. filtering the mass at a temperature of 60°C and drying in a suitable dryer, preferably under vacuum to obtain dried carotenoids crystals. The carotenoids crystals obtained contain Lutein and high amounts of trans- Zeaxanthin (3R,3’R) wherein, preferably the ratio of lutein to trans- zeaxanthin is in the range of 9: 1 to 12:1.

[0061]

[0050] According to an embodiment of the invention the carotenoids crystals are isolated from a plant oleoresin such as non-mutant marigold oleoresin.

[0062]

[0051] According to an embodiment of the invention the alcoholic alkali used in the saponification process could be selected from ethanolic potassium hydroxide and ethanolic sodium hydroxide.

[0063]

[0052] According to an embodiment of the invention the solvent may be selected from the group consisting of isopropyl alcohol, ethyl alcohol, butyl alcohol, iso-amyl alcohol, 1 -propanol, iso propyl acetate, ethyl acetate, butyl acetate, iso butyl acetate, methyl acetate, ethyl acetate, iso-butanol, heptane, pentane and other suitable al cohol / sol vents.

[0053] According to an embodiment of the invention, the resulting carotenoid crystals contain a minimum of 85% carotenoids determined by spectrophotometer of which about 90.0% is all trans-Lutein and about 7.0 to 9.5% all trans-Zeaxanthin along with minute quantities of Beta-carotene and Cryptoxanthin determined by HPLC. Further, the chemical recovery of the final product is in the range of 45 to 70.0% while the physical recovery of the final product is approximately 7 to 12%.

[0064]

[0054] According to an embodiment of the invention, the carotenoid crystals have a purity of at least 85%.

[0065]

[0055] According to an embodiment of the invention, the carotenoids crystals containing high amounts of trans -zeaxanthin are formulated in the form of powder, beadlets, granules, oil dispersion or water dispersion or any other suitable form.

[0066]

[0056] According to an embodiment of the invention, the carotenoids crystals containing high amounts of trans-zeaxanthin (3R,3’R) are used in food and beverages, dietary supplements, nutraceutical or cosmeceutical applications.

[0067]

[0057] While the invention has been described with reference to the explained embodiment, it is not limiting to anybody's skill to make various changes or equivalents without altering or departing from the main scope of this invention. Therefore, it is intended that the invention not be limiting to the embodiment described but will cover and include all other embodiments falling within the scope of the claims made herein.

[0068]

[0058] The following examples are illustrative, but not limiting, of the methods and compositions of the present invention. Other suitable modifications and adaptations of the variables of the conditions and normally encountered in natural products isolation and purification techniques which are obvious to those skilled in the art are within the spirit and scope of the present invention.Example 1

[0069]

[0059] 25 Kgs of Marigold Oleoresin having 144.51gm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 5Kgs of KOH with a purity of 95% and dissolving it in 35 liters of Ethyl alcohol (1:1.4 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 40 liters of demineralized hot water maintained at a temperature of 70°C was added to the reacted mass and the stirring continued for 10 minutes. The diluted mass with carotenoid crystals was then pumped into a filter press to recover the crystals. Around 250 liters of additional hot water was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press and dried at a temperature of around 55°C for 3 hours under normal pressure.

[0070]

[0060] The physical recovery of the final product was 11.68%. The Carotenoid crystals obtained contained 89.05% carotenoids (determined by spectrophotometer) of which 92.61% was all trans-Lutein, 6.12% all trans- Zeaxanthin, 0.10% Beta Carotene and 0.36% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 71.97%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 15:1.

[0071]

[0061] Here, Example 1 is an exemplary process indicating the commonly used process of isolation of carotenoids crystals from marigold oleoresin. It can be seen from this process that the ratio of lutein and zeaxanthin in the obtained crystals is higher i.e., 15 : 1.Example 2

[0072]

[0062] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.4% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40° C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with a purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75° C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Ethyl acetate was added, and the temperature was maintained at 70°C for 30 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 70° C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C for 3 hours under normal pressure.

[0073]

[0063] The physical recovery of the final product was 9.80%. The Carotenoid crystals obtained contained 88.49% carotenoids (determined by spectrophotometer) of which 90.84% was all trans-Lutein, 7.93% all trans- Zeaxanthin, 0.11% Beta Carotene and 0.33% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 60.01%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11.4:1.

[0074] Example 3

[0075]

[0064] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin washomogenized for 10 minutes under stirring at a temperature of 40° C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75° C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Isopropyl acetate were added, and the temperature was maintained at 75°C for 30 minutes. Further, 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75°C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C for 3 hours under normal pressure.

[0076]

[0065] The physical recovery of the final product was 8.40%. The Carotenoid crystals obtained contained 85.29% carotenoids (determined by spectrophotometer) of which 91.01% was all trans-Lutein, 8.21% all trans- Zeaxanthin, 0.05% Beta Carotene and 0.47% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 49.58%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11:1.

[0077] Example 4

[0078]

[0066] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40° C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 litersof Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75° C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Heptane was added, and the temperature was maintained at 75°C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75° C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C for 3 hours.

[0079]

[0067] The physical recovery of the final product was 7.82%. The Carotenoid crystals obtained contained 88.47% carotenoids (determined by spectrophotometer) of which 90.72% was all trans-Lutein, 8.31% all trans- Zeaxanthin, 0.08% Beta Carotene and 0.21% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 47.87%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 10.9:1.

[0080] Example 5

[0081]

[0068] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree ofsaponification to be more than 99% by HPLC, 12.5 liters of 1 -Propanol was added, and the temperature was maintained at 75°C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75°C for another 30 minutes. Further the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C for 3 hours.

[0082]

[0069] The physical recovery of the final product was 10.6%. The Carotenoid crystals obtained contained 88.19% carotenoids (determined by spectrophotometer) of which 91.26% was all trans-Lutein, 7.55% all trans- Zeaxanthin, 0.01% Beta Carotene and 0.33% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 64.69%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 12:1. Example 6

[0083]

[0070] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25 Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of n-Butanol was added, and the temperature was maintained at 75°C for 40 minutes. Further, 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75°C for another 30 minutes.Further the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C to 60°C for 3 hours under normal pressure.

[0084]

[0071] The physical recovery of the final product was 9.02%. The Carotenoid crystals obtained contained 87.49% carotenoids (determined by spectrophotometer) of which 90.75% was all trans-Lutein, 8.14% all trans- Zeaxanthin, 0.23% Beta Carotene and 0.4% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 54.49%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11:1.

[0085] Example 7

[0086]

[0072] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Butyl Acetate was added, and the temperature was maintained at 75°C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75°C for another 30 minutes. Further the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluentto neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 60°C for 3 hours.

[0087]

[0073] The physical recovery of the final product was 8.22%. The Carotenoid crystals obtained contained 87.91% carotenoids (determined by spectrophotometer) of which 90.99% was all trans-Lutein, 8.03% all trans- Zeaxanthin, 0.08% Beta Carotene and 0.28% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 50.0%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11.3:1.

[0088] Example 8

[0089]

[0074] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75° C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Methyl Acetate was added, and the temperature was maintained at 60° C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 60° C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 60°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C for 3 hours under normal pressure.

[0075] The physical recovery of the final product was 7.53%. The Carotenoid crystals obtained contained 88.06% carotenoids (determined by spectrophotometer) of which 90.68% was all trans-Lutein, 8.15% all trans- Zeaxanthin, 0.11% Beta Carotene and 0.33% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 45.88%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11:1.

[0090] Example 9

[0091]

[0076] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40° C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Isobutyl Acetate was added, and the temperature was maintained at 75°C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at the same temperature i.e., 75°C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55°C for 3 hours under normal pressure.

[0092]

[0077] The physical recovery of the final product was 8.2%. The Carotenoid crystals obtained contained 85.24% carotenoids (determined by spectrophotometer) of which90.82% was all trans-Lutein, 7.83% all trans- Zeaxanthin, 0.10% Beta Carotene and 0.33% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 48.37%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11.5:1.

[0093] Example 10

[0094]

[0078] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75° C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Iso Amyl Alcohol was added, and the temperature was maintained at 75°C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75°C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and dried at a temperature of around 55 °C for 3 hours under normal pressure.

[0095]

[0079] The physical recovery of the final product was 7.9%. The Carotenoid crystals obtained contained 85.86% carotenoids (determined by spectrophotometer) of which 90.74% was all trans-Lutein, 8.15% all trans- Zeaxanthin, 0.10% Beta Carotene and 0.32% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 46.94%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11:1.Example 11

[0096]

[0080] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75° C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 12.5 liters of Iso butanol was added, and the temperature was maintained at 75°C for 60 minutes. Further 12.5 liters of Ethyl alcohol was added, and the temperature was maintained at 75° C for another 30 minutes. Further, the mass was pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and tray dried at a temperature around 55°C for 3 hours under normal pressure.

[0097]

[0081] The physical recovery of the final product was 7.56%. The Carotenoid crystals obtained contained 86.10% carotenoids (determined by spectrophotometer) of which 91.28% was all trans-Lutein, 7.71% all trans- Zeaxanthin, 0.10% Beta Carotene and 0.23% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 45.04%. The ratio of all trans-Lutein to all trans- Zeaxanthin was 11.8:1.

[0098] Example 12

[0099]

[0082] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin washomogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 125 liters of demineralized hot water maintained at a temperature of 70° C was added to the reacted mass and the stirring was continued for 30 minutes. The diluted mass with carotenoid crystals was then pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates, mix it in 30 liters of ethyl alcohol and agitate at 65°C for 30 minutes. The mass was filtered under vacuum, the wet crystals were collected and mixed with 25 liters of Iso Amyl Alcohol maintained at a temperature of 75°C for 30 minutes. The mass was filtered at the same temperature and to the wet crystals 25 liters of ethyl alcohol was added while maintaining a temperature of 60° C for 30 minutes. The mass was filtered and the product dried at a temperature of around 55° C for 5-7 hours, preferably under vacuum.

[0100]

[0083] The physical recovery of the final product was 8.5%. The Carotenoid crystals obtained contained 95.76% carotenoids (determined by spectrophotometer) of which 89.89% was all trans-Lutein, 8.27% all trans- Zeaxanthin, 0.005% Beta Carotene and 0.27% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 56.33%. The ratio of all trans Lutein to all trans Zeaxanthin was 10.9:1.

[0101] Example 13

[0084] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40° C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 125 liters of demineralized hot water maintained at a temperature of 70°C was added to the reacted mass and the stirring was continued for 30 minutes. The diluted mass with carotenoid crystals was then pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and mixed with 30 liters of ethyl alcohol and agitated at 65°C for 30 minutes. The mass was filtered under vacuum and the wet crystals were collected and mixed in 25 liters of Heptane maintained at a temperature of 75°C for 30 minutes. The mass was filtered at the same temperature. To the wet crystals was added 25 liters of ethyl alcohol and a temperature of 60°C was maintained for 30 minutes. The mass was filtered, and the product was vacuum dried at a temperature of around 55° C for 5-7 hours.

[0102]

[0085] The physical recovery of the final product was 8.8%. The Carotenoid crystals obtained contained 97.28% carotenoids (determined by spectrophotometer) of which 91.98% was all trans-Lutein, 7.6% all trans- Zeaxanthin, 0.01% Beta Carotene and0.26% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 59.24%. The ratio of all trans Lutein to all trans Zeaxanthin was 12:1. Example 14

[0103]

[0086] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 125 liters of demineralized hot water maintained at a temperature of 70° C was added to the reacted mass and the stirring was continued for 30 minutes. The diluted mass with carotenoid crystals was then pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and 30 liters of ethyl alcohol was mixed in it and agitated at 65° C for 30 minutes. The mass was filtered under vacuum, the wet crystals were collected and mixed with 25 liters of n-Butanol maintained at a temperature of 75°C for 30 minutes. The mass was filtered at the same temperature. To the wet crystals was added 25 liters of ethyl alcohol and maintained at a temperature of 60°C for 30 minutes. The mass was filtered and the product dried at a temperature of around 55°C for 5-7 hours, preferably under vacuum.

[0087] The physical recovery of the final product was 7.01%. The Carotenoid crystals obtained contained 96.87% carotenoids (determined by spectrophotometer) of which 88.77% was all trans-Lutein, 9.14% all trans- Zeaxanthin, 0.05% Beta Carotene and 0.32% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 47.00%. The ratio of all trans Lutein to all trans Zeaxanthin was 9.7:1. Example 15

[0104]

[0088] 25Kgs of Marigold Oleoresin having 144.5 Igm / Kg or 14.45% of Xanthophylls was taken in 100 liters capacity reactor with an agitator. The Oleoresin was homogenized for 10 minutes under stirring at a temperature of 40°C with either steam or hot water in the jacket of the reactor as heating medium. Ethanolic KOH was prepared by taking 6.25Kgs of KOH with purity of 95% and dissolving it in 37.5 liters of Ethyl alcohol (1 : 1.5 volumes). The prepared ethanolic KOH was added into reaction vessel slowly, containing the homogenized mass. The saponification reaction was carried out at a temperature of 75°C for 30 minutes. After ensuring the degree of saponification to be more than 99% by HPLC, 125 liters of demineralized hot water maintained at a temperature of 70°C was added to the reacted mass and the stirring was continued for 30 minutes. The diluted mass with carotenoid crystals was then pumped into a filter press to recover the crystals. Around 250 liters of additional hot water maintained at a temperature of 70°C was pumped through the filter press to wash the unwanted impurities and bring down the pH of the effluent to neutral around 7.0. The wet crystals were then collected from the filter press plates and mixed with 30 liters of ethyl alcohol and agitated at 65°C for 30 minutes. The mass was filtered under vacuum, the wet crystals were collected and mixed with 25 liters of Iso-Butanol maintained at a temperature of 75°C for 30 minutes. The mass was filtered at the same temperature. To the wet crystals was added 25 liters of ethyl alcohol and a temperature of 60°C wasmaintained for 30 minutes. The mass was filtered and the product dried at a temperature of around 55° C for 5-7 hours.

[0105]

[0089] The physical recovery of the final product was 7.1%. The Carotenoid crystals obtained contained 95.99% carotenoids (determined by spectrophotometer) of which 89.07% was all trans-Lutein, 9.78% all trans- Zeaxanthin, 0.01% Beta Carotene and 0.89% Cryptoxanthin (determined by HPLC). The chemical recovery of the final product was 47.1%. The ratio of all trans Lutein to all trans Zeaxanthin was 9.1:1.

Claims

We Claim:

1. Carotenoids crystals comprising of lutein and trans-zeaxanthin (3R, 3 ’R) in a ratio in the range of 9:1 to 12:1 and other carotenoids in minute amounts wherein the carotenoids crystals are derived from non-mutant plant oleoresin, preferably from oleoresin derived from non-mutant marigold flowers.

2. The carotenoids crystals as claimed in claim 1 wherein the said crystals are formulated in the form of powder, beadlets, granules, oil dispersion or water dispersion or any other suitable form.

3. The carotenoids crystals as claimed in claim 1 wherein the said crystals are used in food and beverages, dietary supplements, nutraceutical or cosmeceutical applications.

4. A process for isolating carotenoids crystals as claimed in claim 1 wherein the process comprises of:a) taking a pre-defined amount of a plant oleoresin in a reactor with an agitator and homogenizing the oleoresin for 10 minutes at a temperature of 40°C under stirring;b) adding an alcoholic alkali into the reaction vessel slowly, containing the homogenized mass of oleoresin, at a temperature of 75°C for 30 minutes to saponify the mass and ensuring the degree of saponification to be more than 99% by HPLC,c) subjecting the reactant mass to crystallization by adding a suitable solvent under continuous stirring at a temperature of 60°C - 75°C for 30-60 minutes followed by adding ethyl alcohol under continuous stirring at a temperature of 60°C - 75°C for 30-40 minutes;d) filtering the diluted mass in a filter to recover the crystals and adding hot water at a temperature in the range of 60°C to 70°C, to wash away the unwanted impurities and bring down the pH of the effluent to neutral around 7.0;e) collecting the wet crystals from the filter and drying in a suitable dryer to obtain dried carotenoids crystals;wherein the carotenoids crystals obtained contain Lutein and high amounts of trans-Zeaxanthin (3R,3’R), wherein, preferably the ratio of lutein to t-zeaxanthin is in the range of 9:1 to 12:1.

5. A process for isolating carotenoids crystals as claimed in claim 1 wherein the process comprises of:a) taking a pre-defined amount of a plant oleoresin in a reactor with an agitator and homogenizing for 10 minutes at a temperature of 40°C under stirring; b) adding an alcoholic alkali into the reaction vessel slowly, containing the homogenized mass of oleoresin, at a temperature of 75°C for 30 minutes to saponify the mass and ensuring the degree of saponification to be more than 99% by HPLC;c) subjecting the mass to crystallization by adding demineralized hot water maintained at a temperature of 60°C - 75°C is added to the reacted mass while stirring for 30 minutes;d) filtering the diluted mass with carotenoid crystals is then filtered in a filter to recover the crystals and neutralizing the carotenoids crystals using hot water at a temperature of 60°C to 70°C, to wash away the unwanted impurities and bring down the pH of the effluent to neutral around 7.0;e) filtering the wet crystals, collecting them from the filter and mixing an alcohol at 65°C for 30 minutes, wherein the alcohol may be ethyl alcohol or isopropyl alcohol;f) filtering the mass under vacuum at a temperature of 65°C, collecting the wet crystals and mixing with a solvent maintained at a temperature of 75°C for 30 minutes;g) filtering the mass under vacuum and collecting the wet crystals and adding ethyl alcohol maintained at a temperature of 60°C for 30 minutes; h) filtering the mass at a temperature of 60°C and drying in a suitable dryer, preferably under vacuum to obtain dried carotenoids crystals; wherein the carotenoids crystals obtained contain Lutein and high amounts of trans-Zeaxanthin (3R,3’R), wherein, preferably the ratio of lutein to t-zeaxanthin is in the range of 9:1 to 12:1.

6. The process as claimed in anyone of claims 4 and 5 wherein the alcoholic alkali used in the saponification process is selected from ethanolic potassium hydroxide and ethanolic sodium hydroxide.

7. The process as claimed in claim 4 wherein the solvent in step (c) is selected from the group consisting of isopropyl alcohol, ethyl alcohol, butyl alcohol, iso-amyl alcohol, 1 -propanol, iso propyl acetate, ethyl acetate, butyl acetate, iso butyl acetate, methyl acetate, ethyl acetate, iso-butanol, heptane, pentane and other suitable alcohol / solvents.

8. The process as claimed in claim 5 wherein the solvent in step (f) is selected from the group consisting of isopropyl alcohol, ethyl alcohol, butyl alcohol, iso-amyl alcohol, 1 -propanol, iso propyl acetate, ethyl acetate, butyl acetate, iso butylacetate, methyl acetate, ethyl acetate, iso-butanol, heptane, pentane and other suitable alcohol / solvents.

9. The carotenoids crystals as claimed in claims 1 to 3 and as obtained in the process of claims 4 to 8 wherein carotenoids crystals have a purity of at least 85%.

10. The carotenoids crystals as claimed in claim 9 wherein the carotenoids crystals have a purity in the range of 95% to 97%, preferably.

11. The process as claimed anyone of claims 4 to 10 wherein the resulting carotenoid crystals contain at least 85% carotenoids determined by spectrophotometer of which about 90.0% is all trans-Lutein and about 7.0 to 9.5% all trans-Zeaxanthin along with minute quantities of Beta-carotene and Cryptoxanthin determined by HPLC.