Method for preparing high-purity lutein ester crystal and application thereof

Abstract

The invention relates to a method for preparing a high-purity lutein ester crystal and application thereof. The present disclosure provides a method for preparing lutein esters by providing a raw material selected from marigold flowers or marigold powder; extracting the raw material with ethanol followed by acetone; and concentrating to obtain an extract, which can then be crystallized to obtain lutein ester crystals of high purity. Further, the present disclosure provides a method for preparing lutein esters by providing a raw material selected from marigold flowers or marigold powder; extracting the raw materials by using isopropanol; and concentrating to obtain an extract, which can then be crystallized to obtain lutein ester crystals of high purity.

Description

Technical Field

[0001] The present disclosure relates generally to the field of carotenoids, particularly lutein esters and lutein, and methods for their preparation. The present disclosure particularly relates to methods for preparing lutein ester crystals. Background Art

[0002] The main carotenoids in marigold petals are xanthophylls, with trace amounts of carotenes such as beta-carotene and beta-cryptoxanthin, as well as plant waxes. Marigold petals are a rich source of xanthophylls, the main components of which are lutein esters and, to a lesser extent, zeaxanthin esters. Both lutein and zeaxanthin exist as monoesters and diesters of lauric, myristic, palmitic, and stearic acids. Lutein esters obtained from such sources can be hydrolyzed enzymatically or alkaline to produce free lutein. Lutein is used in feed compositions, as a colorant, and as a nutritional food supplement in foods.

[0003] Recent studies have shown that lutein esters have high or higher bioavailability than free lutein (e.g., see WO1998045241A2). Therefore, there is a need to produce lutein esters as a more natural form of lutein. Current processes for extracting lutein esters include multiple steps with low xanthophyll recoveries, low product yields, and are expensive, involving extraction using solvents such as hexane, tetrahydrofuran, subcritical fluids, or supercritical fluids. Such processes include the use of oleoresin obtained from marigold flowers, which is further processed to obtain lutein esters, making it an energy-intensive and cumbersome process.

[0004] For example, US Pat. No. 4,048,203 A discloses a process involving the use of marigold oleoresin processed with hot alkyl alcohol to obtain about 51% lutein fatty acid ester.

[0005] CN1432567A discloses a method for producing lutein esters from marigold oleoresin by extraction with acetone, wherein the obtained acetone extract is concentrated and crystallized using butanol or isopropanol, however, the yield of the obtained product is low.

[0006] EP1857441B1 discloses a method for separating and purifying xanthophyllfatty acid esters from marigold oleoresin, which method involves multiple extraction steps using an extractant comprising n-hexane. Therefore, there is a need to develop a simple, cost-effective and efficient process for producing high-purity xanthophyllfatty acid esters. Summary of the Invention

[0007] In one aspect of the present disclosure, a method for preparing lutein esters is provided, comprising: (a) providing a raw material selected from shredded marigold flowers, marigold meal, or a combination thereof; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol, or a combination thereof to obtain a polar solvent fraction and a residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; and (d) concentrating the acetone fraction.

[0008] In one aspect of the present disclosure, there is provided a method for preparing lutein esters, the method comprising: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) subjecting the raw material to extraction with an extractant comprising isopropyl alcohol to obtain an isopropyl alcohol fraction; and (d) concentrating the isopropyl alcohol fraction, and optionally subsequently crystallizing to obtain lutein ester crystals.

[0009] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. DETAILED DESCRIPTION

[0010] Those skilled in the art will appreciate that the present disclosure is subject to variations and modifications other than those specifically described. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all such steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of any or more such steps or features.

[0011] definition

[0012] For convenience, before further describing the present disclosure, certain terms used in this specification and examples are described here. These definitions should be read in light of the remainder of the present disclosure and understood by those skilled in the art. The terms used herein have meanings that are generally recognized and known to those skilled in the art. However, for convenience and completeness, specific terms and their meanings are set forth below.

[0013] The articles “a,” “an,” and “the” are used to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0014] Throughout this specification, unless the context requires otherwise, the word "comprise" and variations such as "comprises and comprising" are used in an inclusive, open-ended sense and will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps. It is not intended to be interpreted as "consisting only of."

[0015] As used herein, the term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0016] As used herein, the term "xanthophylls" broadly refers to carotenoids and / or their esters. Examples include lutein, zeaxanthin, lutein esters, zeaxanthin esters, beta carotene, cryptoxanthin, and the like.

[0017] As used herein, the term "lutein" refers to a carotenoid that forms the main component of the xanthophylls present in marigold flowers, particularly petals. As used herein, the term "lutein ester" refers to lutein monoesters and / or diesters, for example, monoesters and / or diesters of lauric acid, myristic acid, palmitic acid, and stearic acid.

[0018] As used herein, the term "zeaxanthin" refers to a carotenoid that forms a minor component of the xanthophylls present in marigold flowers, particularly petals. As used herein, the term "zeaxanthin ester" refers to mono- and / or diesters of zeaxanthin, for example, mono- and / or diesters of lauric, myristic, palmitic, and stearic acids.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0020] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods, as described herein, are clearly within the scope of the present disclosure.

[0021] As discussed in the background, currently known methods involve using marigold oleoresin to prepare lutein esters. Such methods involve the use of extractants such as n-hexane, tetrahydrofuran, and subcritical or supercritical fluids. For pharmaceutical and nutraceutical applications of lutein esters, the use of such solvents may not be preferred. Furthermore, n-hexane is a non-selective solvent that can also extract undesirable lipophilic compounds such as fatty acids and other carotenoids, potentially compromising the purity of lutein ester extracts from marigold flowers. To achieve higher purity, further purification techniques such as enrichment and chromatography are typically required to separate the lutein esters from these co-extracted substances. Therefore, the isolation of lutein esters with high purity and good recovery has always been challenging. Furthermore, compared to n-hexane, alcohol-based solvents, particularly ethanol, that adhere to green chemistry principles are more sustainable and environmentally friendly. Therefore, the inventors of the present invention provide a simple, cost-effective, and efficient process that directly uses marigold flowers or marigold powder as a raw material. According to the method disclosed herein. Thus, the number of processing steps is minimized while achieving high purity lutein ester crystals with good yield and high recovery percentage.The process includes a smaller number of unit operations and has increased product recovery.

[0022] Embodiments herein provide methods for preparing lutein esters. According to embodiments herein, the method can produce lutein esters with high purity. In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol, or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; and (d) concentrating the acetone fraction, or concentrating the acetone fraction to obtain an extract. In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol, or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; (d) concentrating the acetone fraction to obtain an extract comprising lutein esters, followed by crystallization.

[0023] According to embodiments herein, the method for preparing lutein esters comprises using a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof. According to embodiments herein, the raw material can be in the form of chopped marigold flowers, marigold powder, or a combination thereof. Therefore, in one embodiment, the raw material is selected from chopped marigold flowers, marigold powder, or a combination thereof. According to the present disclosure, marigold refers to a plant of the genus Tagetes, in particular Tagetes erecta. In one embodiment, marigold refers to the flower of a plant of the genus Tagetes. Marigold flowers are composed of various natural compounds such as terpenes, xanthophylls, and flavonoids.

[0024] In a preferred embodiment, marigold seeds (originating from seeds in Thailand) are grown under conditions suitable for flowering, and the flowers are then harvested for lutein ester extraction and preparation according to the embodiments herein.

[0025] As used herein, the term "chopped marigold flowers" refers to fresh flowers that are preferably silaged, dejuiced, and shredded. Dejuicing is performed to remove water content from the ensiled flowers, preferably by mechanical pressing. The flowers are ensiled by subjecting the flowers to an ensilage process. As used herein, the term "ensiling" refers to the process of subjecting plant material to anaerobic bacterial fermentation. Ensiled flowers refer to the fermented mass obtained by storing flowers under anaerobic conditions. In a preferred embodiment, the ensiled flowers are obtained by treating the marigold flowers with at least one enzyme selected from cellulases, hemicellulases, cellobiohydrases, pectinases, galactomannanases, proteases, lipases, or combinations thereof, preferably for a period of at least 5 days or about 5 to 10 days. Alternatively, in other embodiments, ensilage can be performed without the use of enzymes. The ensiled flowers can then be dejuiced and shredded to obtain chopped marigold flowers. In one embodiment, fresh marigold flowers are ensiled, dejuiced, and chopped to obtain chopped marigold flowers.

[0026] As used herein, the term "marigold flour" refers to dehydrated biomass derived from marigold flowers. The term includes flour in any form, including but not limited to pellet form, granule form, or powder form.

[0027] In one embodiment, marigold powder is obtained by: ensiling marigold flowers with at least one enzyme selected from the group consisting of cellulase, hemicellulase, cellulose biohydratase, pectinase, galactomannanase, protease, lipase, and combinations thereof, preferably for at least 5 days or about 5 to 10 days; and dehydrating to obtain marigold powder. Preferably, the ensiled flowers are further dejuiced to remove moisture and minced / ground, followed by drying under controlled conditions such that the marigold powder has a moisture content in the range of 8 to 12 wt%. Preferably, the marigold powder is in the form of a powder. Therefore, in one embodiment, marigold powder is obtained by: ensiling marigold flowers with at least one enzyme selected from the group consisting of cellulase, hemicellulase, cellulose biohydratase, pectinase, galactomannanase, protease, lipase, and combinations thereof, preferably for at least 5 days or about 5 to 10 days; dejuicing; mincing; and dehydrating to obtain marigold powder. Marigold powder can be further processed into other forms, such as pellets to obtain marigold pellets suitable for long-term storage and high-throughput processing. Mincing can be performed using, for example, a mechanical chopper, a compressor, etc. by methods generally known in the art.

[0028] In one embodiment, marigold flour is obtained by a process comprising: ensilaging marigold flowers with at least one enzyme selected from the group consisting of cellulase, hemicellulase, cellulose biohydratase, pectinase, galactomannanase, protease, lipase, and combinations thereof, preferably for a period of at least 5 days or about 5 to 10 days; and dehydrating to obtain marigold flour.

[0029] In another embodiment, marigold powder is obtained by a process comprising: ensilaging marigold flowers with at least one enzyme selected from the group consisting of cellulase, hemicellulase, cellulose biohydratase, pectinase, galactomannanase, protease, lipase, and combinations thereof, preferably for a period of at least 5 days or about 5 to 10 days; dejuicing; chopping; and dehydrating to obtain marigold powder.

[0030] In one embodiment, the chopped marigold flowers are obtained by a process comprising: ensilaging the marigold flowers for a period of preferably at least 5 days or about 5 to 10 days using at least one enzyme selected from the group consisting of cellulase, hemicellulase, cellulose biohydratase, pectinase, galactomannanase, protease, lipase, and combinations thereof; dejuicing; and chopping to obtain chopped marigold flowers.

[0031] In one embodiment, a method for preparing lutein esters comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol, or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; and (d) concentrating the acetone fraction.

[0032] In one embodiment, the method for preparing lutein esters comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol, or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; and (d) concentrating the acetone fraction to obtain an extract comprising lutein esters, followed by crystallization to obtain lutein ester crystals.

[0033] In one embodiment, a method for preparing lutein esters comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol, or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction comprising luteins; and (d) concentrating the acetone fraction to obtain an extract comprising lutein esters, and crystallizing the extract to obtain lutein ester crystals.

[0034] The step of subjecting the raw material to extraction in methods according to embodiments herein comprises extracting the raw material using a polar solvent.

[0035] As used herein, the term "extraction" or "extracting" refers to solvent extraction that achieves the transfer of a substance from one phase to another. The phase can be solid or liquid, and therefore, the extraction can be a liquid-liquid extraction or a solid-liquid extraction. The extraction process can be performed once or multiple times. The extraction can be performed in a solvent extractor. In one embodiment, the extraction step is repeated 1 to 5 times using fresh solvent each time. In one embodiment, the extraction step is repeated 1, 2, 3, 4, 5 or more times, preferably 2 to 5 times, using fresh solvent.

[0036] In one embodiment, the raw material is subjected to extraction with at least one polar solvent, preferably selected from ethanol, methanol or a combination thereof.

[0037] In one embodiment, the ratio of raw materials to polar solvent is in the range of 1:2 to 1:8, or 1:3 to 1:6, preferably 1:4, 1:5, or 1:6. In one embodiment, the ratio of chopped marigold flowers to polar solvent is in the range of 1:2 to 1:8, or 1:3 to 1:6, preferably 1:4, 1:5, or 1:6. In one embodiment, the ratio of marigold powder to polar solvent is in the range of 1:2 to 1:8, or 1:3 to 1:6, preferably 1:4, 1:5, or 1:6.

[0038] In one embodiment, extraction with a polar solvent is carried out at a temperature in the range of 30 to 60°C, preferably 40 to 50°C, with continuous circulation of the solvent for a period of 20 to 100 minutes, preferably 20 to 60 minutes or 20 to 40 minutes, more preferably 30 minutes.

[0039] In another embodiment, the extraction using a polar solvent is repeated 2 to 5 times to obtain multiple fractions, which can then be combined. In the method according to the embodiments of the present invention, the extraction using a polar solvent obtains a polar solvent fraction and residual biomass. As used herein, the term "polar solvent fraction" or "polar solvent miscella" is the fraction of the solvent mixture leaving the solvent extractor. In one embodiment, the polar solvent fraction is obtained after extraction. According to embodiments of the present invention, the polar solvent is an ethanol fraction containing polyphenols such as quercetin. In one embodiment, the polar solvent fraction contains at least one polyphenol selected from quercetin, 6-hydroxykaempferol, quercetin, tagetin, or a mixture thereof.

[0040] As used herein, the term "residual biomass" refers to the solid mass obtained after removal of the polar solvent fraction. In one embodiment, the residual biomass is obtained as a residue after extraction.

[0041] In one embodiment, the residual biomass is extracted with an extractant comprising acetone to obtain an acetone fraction. In one embodiment, the acetone fraction comprises xanthophylls. In one embodiment, the acetone fraction comprises xanthophylls including lutein esters and zeaxanthin esters. The extraction using the extractant comprising acetone can be repeated, preferably 1 to 5 times, to achieve maximum transfer of xanthophylls from the residual biomass to the acetone fraction. In one embodiment, the extraction using the extractant comprising acetone can be carried out 1 to 5 times, or 1 to 4 times, preferably 4 times, using fresh extractant each time. In one embodiment, the extractant is acetone or an acetone solution.

[0042] In one embodiment, the ratio of residual biomass to extractant is in the range of 1:2 to 1:8, preferably 1:5.

[0043] In one embodiment, a method is provided wherein extracting the residual biomass with the extractant is carried out at a temperature in the range of 25 to 52° C., preferably 30 to 45° C. or 40 to 45° C., with continuous circulation of the extractant for a period of 20 to 60 minutes, preferably 20 to 40 minutes, more preferably 30 minutes. In another embodiment, a method is provided wherein extracting the residual biomass with the extractant is carried out at a temperature in the range of 40 to 45° C., with continuous circulation of an extractant comprising acetone for a period of 20 to 40 minutes, preferably 30 minutes.

[0044] In another embodiment, the extraction with acetone is repeated 2 to 5 times to obtain multiple fractions, which can then be combined. In the method according to the embodiments herein, the extraction with acetone obtains an acetone fraction. As used herein, the term "acetone fraction" or "acetone mixed oil" refers to the fraction with acetone that exits the extractor and contains xanthophylls.

[0045] According to embodiments of the present invention, the acetone fraction is further concentrated. In one embodiment, a method is provided, which includes concentrating the acetone fraction. In another embodiment, a method is provided, which includes concentrating the acetone fraction to obtain an extract or an extract comprising lutein esters. The acetone fraction can be concentrated to remove the solvent completely (i.e., desolvation), or until the extract comprises 1 to 12 wt%, 1 to 10 wt%, 1 to 5 wt%, 5 to 12 wt%, or 5 to 10 wt% extractive.

[0046] In another embodiment, the method comprises concentrating the acetone fraction, wherein the concentration is performed to obtain an extract comprising 1 to 12 wt%, 1 to 10 wt%, 1 to 5 wt%, 5 to 12 wt%, or 5 to 10 wt% extractables.

[0047] In one embodiment, the process comprises concentrating the acetone fraction, wherein the concentration is performed to obtain an extract comprising 1 to 12 wt% extractables, preferably 1 to 5% extractables when the starting material is chopped marigold flowers, preferably 5 to 10% extractables when the starting material is marigold powder.

[0048] In one embodiment, the acetone fraction is concentrated until the extract comprises 1 to 12 wt%, 1 to 10 wt%, 1 to 5 wt%, 5 to 12 wt%, or 5 to 10 wt% extractables.

[0049] In one embodiment, the method comprises concentrating the acetone fraction to remove the solvent (i.e., acetone) or for desolventizing, and obtaining an extract. In one embodiment, the extract obtained after desolventizing or concentrating the acetone fraction to remove the solvent is an oleoresin. In one embodiment, the method comprises concentrating the acetone fraction, wherein the concentration is performed to obtain an extract comprising xanthophylls in a weight range of 15 to 30 wt%, lutein in a weight range of 70 to 80 wt% of the xanthophylls, and zeaxanthin in a weight range of 3 to 6 wt%. In one embodiment, the extract is an oleoresin comprising xanthophylls in a weight range of 15 to 30 wt%, lutein in a weight range of 70 to 80 wt% of the xanthophylls, and zeaxanthin in a weight range of 3 to 6 wt%, wherein the yield of the oleoresin is in a range of 5 to 10 wt%, or 6 to 10 wt% relative to the raw material.

[0050] Concentration can be carried out by methods well known in the art. For example, the extract can be concentrated using a vacuum evaporator. According to the present disclosure, extract refers to the component transferred from the residual biomass to the acetone phase. The acetone fraction is concentrated using methods known in the art to obtain an extract rich in extract.

[0051] In one embodiment, the extract comprises 1 to 12% extractables. In another embodiment, the extract comprises 5 to 12% or 5 to 10% extractables. In another embodiment, the extract comprises 1 to 5% extractables. In another embodiment, the extract comprises 8 to 10% extractables.

[0052] In one embodiment, when the starting material is chopped marigold flowers, the extract comprises 1 to 5% extractables. In one embodiment, when the starting material is marigold powder, the extract comprises 5 to 10% extractables.

[0053] In one embodiment, the method comprises subjecting the extract to crystallization to obtain crystals comprising lutein esters, and a mother liquor. In one embodiment, crystallization is performed to obtain crystals comprising lutein esters, and a mother liquor.

[0054] In one embodiment, the crystallization is carried out at a temperature in the range of 7 to 40° C. for a period of 8 to 60 hours, 8 to 50 hours, 10 to 50 hours, 10 to 40 hours, 10 to 30 hours, 22 to 50 hours, or 20 to 50 hours, preferably selected from 10 hours, 12 hours, 48 hours, or 60 hours. In one embodiment, the crystallization is carried out at a temperature in the range of 7 to 40° C., 9 to 37° C., 10 to 37° C., 20 to 37° C., 7 to 20° C., or 30 to 37° C.

[0055] In another embodiment, the crystallization is carried out at a temperature in the range of 9 to 37° C. for a period of 20 to 50 hours, preferably 48 hours. In yet another embodiment, the crystallization is carried out at a temperature in the range of 20 to 35° C. for a period of 20 to 50 hours, preferably 48 hours. In yet another embodiment, the crystallization is carried out in a continuous stirred tank reactor (CSTR) at a temperature in the range of 30 to 37° C. for a period of 22 to 50 hours, preferably 48 hours.

[0056] In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder or a combination thereof, wherein the raw material is marigold powder; (b) extracting the raw material with at least one polar solvent selected from ethanol, methanol or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; and (d) concentrating the acetone fraction to obtain an extract comprising lutein esters, followed by crystallization to obtain lutein ester crystals, wherein the crystallization is carried out at a temperature in the range of 7 to 40°C, preferably 10 to 37°C or 20 to 37°C, for a time of 20 to 50 hours, preferably 48 hours.

[0057] In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder or a combination thereof, wherein the raw material is chopped marigold flowers; (b) subjecting the raw material to extraction with at least one polar solvent selected from ethanol, methanol or a combination thereof to obtain a polar solvent fraction and residual biomass; (c) subjecting the residual biomass to extraction with an extractant comprising acetone to obtain an acetone fraction; and (d) concentrating the acetone fraction to obtain an extract comprising lutein esters, and subjecting the extract to crystallization to obtain lutein ester crystals, wherein the crystallization is carried out at a temperature in the range of 7 to 40° C., preferably 10 to 37° C., for a period of 10 to 50 hours or 20 to 50 hours, preferably 10, 12 or 24 hours. In one embodiment, a method is provided, wherein the extraction steps of step (b) and / or step (c) are repeated at least once, preferably 2 to 5 times, preferably until complete extraction is achieved.

[0058] According to the embodiments of the present invention, the lutein ester crystals are further filtered and dried to obtain lutein ester crystals. Filtration and drying can be performed using methods generally known in the art. Drying is preferably performed using a dehumidified air dryer (DHAD) at a temperature in the range of 25 to 50° C., 45 to 48° C., or 45 to 48° C. In one embodiment, a method is provided, wherein the method comprises filtering and drying to obtain lutein ester crystals, wherein drying is performed at a temperature in the range of 25 to 50° C., 45 to 48° C., or 45 to 48° C.

[0059] In one embodiment, the yield of the lutein esters is in the range of 0.25 to 2.5 wt% relative to the raw material. In one embodiment, the yield of the lutein esters is in the range of 1.1 to 2.5 wt% relative to marigold powder. In another embodiment, the yield of the lutein esters is in the range of 2.1 to 2.5 wt% relative to marigold powder. In one embodiment, the yield of the lutein esters is in the range of 0.25 to 1.0 wt% relative to chopped marigold flowers.

[0060] In one embodiment, the yield of the lutein ester crystals is in the range of 0.25 to 2.5 wt % relative to the raw material, preferably 1.1 to 2.5 wt % when the raw material is marigold powder, or preferably 0.25 to 1.0 wt % when the raw material is chopped marigold flowers.

[0061] In one embodiment, the purity of lutein ester is at least 55wt%. In one embodiment, the purity of lutein ester is at least 55wt%, at least 58wt%, at least 60wt%, at least 62wt%, at least 64wt%, at least 66wt%, at least 68wt%, at least 70wt%, at least 72wt%, at least 74wt%, at least 76wt%, at least 78wt%, at least 80wt%, at least 82wt%, at least 84wt%, at least 86wt%, at least 87wt%, or at least 88wt%. In one embodiment, the purity of lutein ester is in the range of 55 to 62wt%. In another embodiment, the purity of lutein ester is in the range of 70 to 88wt% or 75 to 88wt%. In yet another embodiment, a method is provided wherein the raw material is marigold powder and the purity of the lutein ester is in the range of 84 to 88wt%. In yet another embodiment, a method is provided wherein the raw material is marigold powder and the purity of the lutein ester is in the range of 75 to 88 wt%.

[0062] In one embodiment, the purity of the lutein ester is in the range of 70 to 88 wt%, and wherein the crystals further comprise zeaxanthin esters in the range of 2 to 6 wt%. In another embodiment, the purity of the lutein ester is in the range of 84 to 88 wt%, and wherein the crystals further comprise zeaxanthin esters in the range of 3 to 6 wt%.

[0063] According to embodiments herein, the mother liquor is further concentrated to obtain an oleoresin. In one embodiment, the oleoresin is saponified to obtain free lutein having a purity of at least 74%. As used herein, the term "saponification" refers to the hydrolysis of esters, e.g., the hydrolysis of lutein esters into free lutein and potassium salts of fatty acids. Saponification can be performed in the presence of a base.

[0064] In one embodiment, the process further comprises concentrating the mother liquor to obtain an oleoresin, wherein the oleoresin comprises xanthophylls in an amount of at least 12%, or 12 to 16 wt%, wherein lutein is in the range of 70 to 75 wt% and zeaxanthin is in the range of 3 to 6 wt%.

[0065] According to embodiments herein, the polar solvent fraction obtained by the method can be further purified using carbon in ethanol. In one embodiment, the method further comprises subjecting the polar solvent fraction to carbon purification in ethanol to obtain quercetin. In one embodiment, a method is provided wherein the quercetin is at least 90% pure.

[0066] The embodiments herein obtain lutein ester crystals with high purity and reduced or no impurities. As used herein, the term impurity includes at least one component selected from the following: protein, ash, pesticides, polycyclic aromatic hydrocarbons (PAH), benzopyrene, pesticides, furans, dioxins, melamine, ethylene oxide, and nitrosamines. In one embodiment, a method is provided, wherein the crystals further include protein in the weight range of 0 to 0.5%, ash in the weight range of 0 to 0.2%, polycyclic aromatic hydrocarbons (PAH) in the amount of 0 to 2ppb, benzopyrene in the amount of 0 to 0.5ppb, pesticides in the amount of 0 to 0.01ppm, dioxins in the amount of 0 to 0.5pg / g, melamine in the amount of 0 to 0.1mg / kg, ethylene oxide in the amount of 0 to 10ppb, and nitrosamines in the amount of 0 to 0.01mg / kg.

[0067] In one embodiment, a method is provided wherein the crystals have an impurity profile comprising 0 to 0.5% protein, 0 to 0.2% ash, 0 to 2 ppb polycyclic aromatic hydrocarbons (PAHs), 0 to 0.5 ppb benzopyrene, 0 to 0.01 ppm pesticides, 0 to 0.5 pg / g dioxins, 0 to 0.1 mg / kg melamine, 0 to 10 ppb ethylene oxide, and 0 to 0.01 mg / kg nitrosamines.

[0068] Embodiments herein further provide a method for preparing lutein esters using an extractant comprising isopropyl alcohol. In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with an extractant comprising isopropyl alcohol to obtain an isopropyl alcohol fraction; and (c) concentrating the isopropyl alcohol fraction and optionally subsequently crystallizing to obtain lutein ester crystals.

[0069] In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) extracting the raw material with an extractant comprising isopropyl alcohol to obtain an isopropyl alcohol fraction; and (c) concentrating the isopropyl alcohol fraction to obtain an extract and subsequently crystallizing to obtain lutein ester crystals.

[0070] In one embodiment, the method comprises: (a) providing a raw material selected from chopped marigold flowers, marigold powder, or a combination thereof; (b) subjecting the raw material to extraction with an extractant comprising isopropyl alcohol to obtain an isopropyl alcohol fraction; and (c) concentrating the isopropyl alcohol fraction followed by crystallization. In one embodiment, a method is provided wherein the extraction step of step (b) is repeated at least once, preferably 2 to 5 times, preferably until complete extraction is achieved.

[0071] In one embodiment, the ratio of raw material to extractant is in the range of 1:2 to 1:8, preferably 1:5. In one embodiment, the ratio of chopped marigold flowers to extractant is in the range of 1:2 to 1:8, preferably 1:5. In one embodiment, the ratio of marigold powder to extractant is in the range of 1:2 to 1:8, preferably 1:5.

[0072] In one embodiment, the raw material is extracted using an extractant comprising isopropanol at a temperature in the range of 50 to 60°C, 53 to 60°C, 53 to 58°C, or 54 to 58°C, preferably at a temperature of 55°C, with the extractant continuously circulated for a period of 20 to 40 minutes, preferably 30 minutes.

[0073] In another embodiment, the extraction using the extractant is repeated 2 to 5 times to obtain multiple fractions, which can then be combined. In the method according to the embodiments of this paper, the extraction using the extractant comprising isopropyl alcohol has obtained the isopropyl alcohol fraction. As used herein, the term "isopropyl alcohol fraction" or "isopropyl alcohol mixed oil" is the fraction of the isopropyl alcohol solvent mixture leaving the solvent extractor. In one embodiment, the isopropyl alcohol fraction is obtained after extraction. According to the embodiments of this paper, the isopropyl alcohol fraction is further concentrated using known methods to obtain an extract comprising 1 to 12 wt%, 5 to 12% or 5 to 10 wt% extract. For example, a continuous vacuum evaporator can be used to concentrate the isopropyl alcohol fraction to obtain the extract.

[0074] In one embodiment, the extract comprises 1 to 12%, 5 to 12%, or 5 to 10% extractables. In another embodiment, the extract comprises 8% to 10% extractables.

[0075] In one embodiment, the method comprises concentrating the isopropanol fraction, wherein the concentrating is performed to obtain an extract comprising 1 to 12%, 5 to 12%, 5 to 10%, or 8 to 10% extractables. Alternatively, the isopropanol fraction can be concentrated to remove the solvent.

[0076] In one embodiment, the method further comprises subjecting the extract to crystallization to obtain crystals comprising lutein esters, and a mother liquor. In one embodiment, crystallization is performed to obtain crystals comprising lutein esters, and a mother liquor.

[0077] In one embodiment, crystallization is carried out at a temperature in the range of 10 to 55° C., 10 to 45° C., 20 to 40° C., 30 to 40° C. for a period of 5 to 90 hours, 20 to 80 hours, 20 to 75 hours, preferably 48, 72, or 24 hours, preferably 48 hours. In another embodiment, crystallization is carried out at a temperature in the range of 30 to 40° C. for a period of 5 to 90 hours, 20 to 80 hours, 20 to 75 hours, preferably 48, 72, or 24 hours. In yet another embodiment, crystallization is carried out at a temperature in the range of 20 to 35° C. for a period of 24 to 72 hours, preferably 48 hours. In yet another embodiment, crystallization is carried out at a temperature in the range of 33 to 35° C. for a period of 24 to 72 hours, preferably 48 hours.

[0078] In one embodiment, the yield of the lutein esters is in the range of 2 to 3 wt% relative to marigold powder. In another embodiment, the yield of the lutein esters is in the range of 2.5 to 3 wt% relative to marigold powder.

[0079] In one embodiment, the purity of the lutein ester is at least 55 wt%. In one embodiment, the purity of the lutein ester is at least 55 wt%, at least 58 wt%, at least 60 wt%, at least 62 wt%, at least 64 wt%, at least 66 wt%, at least 68 wt%, at least 70 wt%, at least 72 wt%, at least 74 wt%, at least 76 wt%, or at least 87 wt%. In one embodiment, the purity of the lutein ester is in the range of 55 to 62 wt%. In one embodiment, the purity of the lutein ester is in the range of 70 to 75 wt%, and wherein the crystals further contain zeaxanthin esters in the range of 2 to 6 wt%. In another embodiment, the purity of the lutein ester is in the range of 72 to 75 wt%, and wherein the crystals further contain zeaxanthin esters in the range of 3 to 6 wt%.

[0080] In one embodiment, the method further comprises concentrating the mother liquor to obtain the oleoresin, wherein the oleoresin comprises xanthophylls in the range of 5 to 8 wt %, wherein lutein is in the range of 70 to 75 wt % and zeaxanthin is in the range of 3 to 6 wt %.

[0081] Although the subject matter has been described in considerable detail with reference to certain embodiments and implementations thereof, other implementations are possible.

[0082] Example

[0083] The disclosure will now be described with working examples, which are intended to illustrate the work of the disclosure and are not intended to restrictively imply any limitation to the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the disclosure pertains. Although methods and materials similar to or equal to those described herein can be used in the practice of disclosed methods and compositions, exemplary methods, devices, and materials are described herein. It should be understood that the disclosure is not limited to described ad hoc methods and experimental conditions, because such methods and conditions can be applied.

[0084] Material

[0085] Marigold flowers are obtained from plants grown from seeds sourced from Thailand.

[0086] Solvents used in this disclosure, such as ethanol, were purchased from Godavari biorefineries Ltd, Karnataka, India; acetone was purchased from Hindustan Organic Chemicals Ltd., Cochin, India; and isopropyl alcohol was purchased from Karnataka Chemical Industries, Bengaluru, India.

[0087] Example 1: Preparation of marigold powder for lutein ester extraction

[0088] Enzyme-assisted silage

[0089] Fresh marigold flowers, having a xanthophylls content of 0.04 to 0.3% by weight, a polyphenol and wax content of 8 to 11% by weight, and a moisture content of 88 to 92% by weight, are mixed with an optimized combination of enzymes, including cellulases, hemicellulases, cellulose biohydratases, pectinases, galactomannanases, proteases, and lipases. The enzyme mixture is dosed at a dose of 0.15% based on the weight of the flowers. The flower biomass is simultaneously ensiled and hydrolyzed for approximately eight days to produce enzyme-ensilaged biomass.

[0090] The ensiled biomass was dejuiced to remove moisture, then chopped and dried to obtain marigold powder. The marigold powder contained xanthophylls ranging from 1.8 to 3.0% relative to the marigold powder as determined by UV analysis, and lutein relative to the xanthophylls content ranging from 75% to 84% (Table 1), as determined by high performance liquid chromatography (HPLC) analysis.

[0091] Table 1

[0092]

[0093] Example 2: Preparation of extract from marigold powder.

[0094] 250g of marigold powder (prepared using Example 1) was placed in an extractor and treated with ethanol at a weight ratio of marigold powder to ethanol (1:4). The mixture was heated at 45°C with stirring and extracted with a specified volume of ethanol. The mixture was then filtered and the residue was re-extracted with ethanol until the ethanol solubles were completely extracted, performing four extraction cycles. The residual biomass was then treated with acetone at a weight ratio of residual biomass to acetone (1:5) at 40°C. Repeated extractions were performed with acetone to recover all xanthophylls from the biomass. The acetone mixed oil was then concentrated to remove the solvent and produce an extract. The extract was tested for its lutein, lutein, and zeaxanthin content. Table 1.1 shows the yield of the extract, the lutein content, and the lutein content and zeaxanthin content in the lutein.

[0095] Table 1.1

[0096] experiment Yield of extract (%) Lutein% Lutein% Zeaxanthin% 1 8.4 22 76.2 4.6 2 8.6 21.3 75.8 4.8

[0097] Extraction of Lutein Esters from Marigold Powder Using Ethanol-Acetone

[0098] Marigold powder (250 g) (prepared using Example 1 of the present disclosure) was placed in a 5 L conical flask and treated with ethanol (polar solvent) in a weight ratio of marigold powder to ethanol ranging from 1:2 to 1:8, as shown in Experiments 1 to 3 in Table 1.2. The mixture was heated at 45°C while stirring and continuously circulating ethanol (polar solvent) for a period of 30 minutes. The mixture was then filtered and the residue was re-extracted with ethanol until the ethanol (polar solvent) soluble contents such as polyphenols were completely extracted, preferably for 4 extraction cycles. The ethanol (polar solvent) fraction was then filtered and the ethanol evaporated. The ethanol (polar solvent) fraction was then evaporated, carbon treated and crystallized to obtain quercetin, which is the main flavonoid (polyphenol) present in marigold flowers. The polyphenol content was determined using UV analysis and the quercetin content was determined using HPLC analysis. The yield of polyphenols was calculated using the following formula:

[0099]

[0100] The residual biomass was extracted with acetone at a weight ratio of residual biomass to acetone ranging from 1:2 to 1:8, as shown in Table 1.2. The mixture was preferably heated to a temperature within the range of 45°C with acetone continuously circulating for 30 minutes. Repeated extractions with acetone were performed to recover all xanthophylls from the meal. The acetone oil mixture was then concentrated to obtain an extract, which was concentrated until 5% to 10% extractables were present in the extract. The extract contained lutein esters, as shown in Table 1.2. The extract was then crystallized at 35°C for 24 to 72 hours, preferably 48 hours, as shown in Experiments 4 to 8 of Table 1.2. The crystals of trans-lutein esters were allowed to settle, filtered, and dried in a dehumidified air dryer (DHAD) at 46°C. Total carotenoids were estimated by UV-Vis spectrophotometry, and the lutein ester content was measured by normal phase HPLC after saponification using methanolic KOH at 56°C for 1 hour. HPLC was performed using hexane / ethyl acetate (65:35) as the mobile phase and a Phenomenex Luna C18 column at a wavelength of 446 nm. The yield of lutein esters was calculated using the following formula:

[0101]

[0102] Similarly, ethanol-acetone extraction can be used on marigold flowers in the wet stage, ensiled flowers that have not been dehydrated (to avoid drying). The use of an alcohol such as ethanol can dehydrate the wet flowers while extracting water-soluble substances and polyphenols. This alcohol-treated biomass can be used for acetone extraction and crystallization of lutein esters. Table 1.2 shows working and non-working examples, where experiments 1 to 9 achieved satisfactory results. However, in experiments 10, 11, and 12, no lutein crystals or oleoresin properties were observed.

[0103] Observations and results

[0104] The extraction method using ethanol and acetone produced lutein ester crystals ranging from 1.1 to 2.5% of the powder, with the purity of trans-lutein ester in the isolated crystals ranging from 84 to 88%, and the crystals further containing 2 to 6% trans-zeaxanthin ester (Table 1.2). In particular, Experiment No. 6, which involved using a 1:4 (i.e., powder:ethanol, weight ratio) and a 1:5 (i.e., powder:acetone, weight ratio) crystallization at 35°C for 48 hours, produced 87% lutein ester crystals.

[0105] The ethanol (polar solvent) fraction yielded a 22 wt% polyphenol product containing 20% pure quercetin. This 20% pure quercetin was further purified to obtain quercetin with a purity greater than 90% (Table 1.3). The by-product obtained by desolventizing the mother liquor (removing acetone) exhibited qualities similar to those of conventional (hexane-extracted) oleoresin. The yield of the by-product formed was 4 to 6% oleoresin relative to the powder, containing 12 to 16 wt% xanthophylls. As shown in Table 1.4, the xanthophylls contained 70 to 75% trans-lutein relative to the lutein. The oleoresin was saponified to obtain free lutein with a purity greater than 74%.

[0106] Table 1.2: Experimental conditions for obtaining lutein esters using the ethanol-acetone extraction method

[0107]

[0108] Table 1.3: Ethanol (polar solvent) fractions

[0109] % Yield of ethanol fraction Polyphenols% Quercetin% 22 30 20

[0110] Table 1.4: Products from mother liquor

[0111] Oil resin yield% Lutein% Lutein% Zeaxanthin% 6 15 75 4.8

[0112] Example 2.1: Large-scale preparation of high-purity crystalline lutein from marigold powder using ethanol-acetone extraction method ester

[0113] 100 kg of marigold powder was placed in an extractor and treated with ethanol at a weight ratio of marigold powder to ethanol (1:4). The mixture was heated at 45°C with stirring and extracted with a specified volume of ethanol. The mixture was then filtered and the residue was re-extracted with ethanol until the ethanol solubles were completely extracted, performing four extraction cycles. The residual biomass was then treated with acetone at a weight ratio of residual biomass to acetone (1:5) at 40°C. Repeated extractions were performed with acetone to recover all xanthophylls from the biomass. The acetone mixed oil was concentrated to obtain an extract with 10% extract and kept at 35°C for 48 hours for crystallization. The settled crystals of trans-lutein esters were filtered out and dried under DHAD at 45 to 48°C. The filtered mixed oil (mother liquor) was desolventized to produce an oleoresin.

[0114] Table 2.1.1: Product spectrum of high-purity crystalline lutein esters from marigold powder

[0115] experiment % Yield of Lutein Esters from Flour % Total Carotenoids % Lutein Esters 1 2.1 91 85.1 2 2.4 92 86 3 2.2 91 85.5

[0116] Common impurities (pollutants) in marigold products are polycyclic aromatic hydrocarbons, dioxins, nitrosamines, ethylene oxide, etc.

[0117] Table 2.1.2: Impurity profile of high-purity crystalline lutein esters

[0118] parameter Experiment 1 Experiment 2 Experiment 3 protein% 0.4 0.41 <0.1 Ash % <0.1 <0.1 <0.1 PAH (polycyclic aromatic hydrocarbons) (ug / kg) Not detected Not detected Not detected Benzopyrene (ug / kg) Not detected Not detected Not detected insecticides Not detected Not detected Not detected Dioxins, furans (pg / g) 0.495 0.451 0.477 Melamine (mg / kg) <0.1 <0.1 <0.1 Ethylene oxide (ppb) <10 <10 <10 Nitrosamines (mg / kg) <0.01 <0.01 <0.01

[0119] Ethanol extract (i.e. polar solvent fraction)

[0120] The ethanolic oil mixture from the experiment was filtered and evaporated to produce an extract with polyphenols as active ingredients, with quercetin being the main polyphenol.

[0121] Table 2.1.3: Ethanol (polar solvent) fractions

[0122] % Yield of ethanol extract % polyphenols % Quercetin Experiment 1 20 30 21 Experiment 2 21 32 22 Experiment 3 18 31 20

[0123] Isolation of quercetin from the ethanol extract (i.e., polar solvent fraction)

[0124] Carbon purification of the ethanol extract in an ethanol medium yielded quercetin with >90% purity.

[0125] Table 2.1.4: Isolation of quercetin

[0126] %Yield % polyphenols % Quercetin Experiment 1 3 92 91 Experiment 2 3.3 91 90.8 Experiment 3 3.5 92 91.2

[0127] Marigold oleoresin from the mother liquor after crystallization of lutein esters

[0128] The by-product obtained by desolventizing the mother liquor was similar in quality to the conventional (hexane extracted) oleoresin, as shown in Table 2.1.5. Saponification of the oleoresin gave free xanthophylls with a minimum purity of 74%.

[0129] Table 2.1.5: Marigold oleoresin from lutein ester by-products

[0130] % oleoresin yield % Luteins % Lutein by HPLC % Zeaxanthin by HPLC Experiment 1 5 14 75 4.2 Experiment 2 4.8 15.2 73 4.6 Experiment 3 5.1 14.3 72 4.1

[0131] Example 2.2: Large-scale preparation of high-purity crystalline lutein esters from marigold powder using ethanol-acetone

[0132] 100 kg of marigold powder was placed in an extractor and treated with ethanol in a weight ratio of marigold powder to ethanol (1:4). The mixture was heated at 40 to 50°C with stirring and extracted with a specified volume of ethanol. The mixture was then filtered and the residue was re-extracted with ethanol until the ethanol solubles were completely extracted, performing 4 extraction cycles. The residual biomass was treated with acetone at a weight ratio of residual biomass to acetone (1:5) at 40 to 45°C. Repeated extractions were performed with acetone to recover all xanthophylls from the biomass. The acetone mixed oil was concentrated to a concentration of 10% and kept at 25°C for 60 hours for crystallization. The settled crystals of trans-lutein esters were filtered out and dried under DHAD at 45 to 48°C.

[0133] The filtered mixed oil (mother liquor) was desolventized to obtain oleoresin marigold.

[0134] Table 2.2.1: Product spectrum of crystalline lutein esters from marigold powder

[0135] experiment Product yield % from powder Total carotenoids% Lutein esters% 1 3.0 62 58 2 3.2 64 59.8 3 2.9 66 61.7

[0136] Table 2.2.2: Impurity profile of crystalline lutein esters

[0137] parameter Experiment 1 Experiment 2 Experiment 3 protein% <0.1 <0.1 <0.1 Ash % <0.1 <0.1 <0.1 PAHs (ug / kg) Not detected Not detected Not detected Benzopyrene (ug / kg) Not detected Not detected Not detected insecticides Not detected Not detected Not detected Dioxins, furans (pg / g) 0.482 0.484 0.478 Melamine (mg / kg) <0.1 <0.1 <0.1 Ethylene oxide (ppb) <10 <10 <10 Nitrosamines (mg / kg) <0.01 <0.01 <0.01

[0138] Polyphenols from the ethanol extract (i.e., polar solvent fraction)

[0139] The ethanolic oil mixture from the experiments was filtered and evaporated to yield an extract with polyphenols as active ingredients.

[0140] Table 2.2.3: Ethanol Extracts

[0141]

[0142] Isolation of quercetin from ethanol extract

[0143] Quercetin was isolated from the ethanol extract with >90% purity by carbon treatment in an ethanol medium.

[0144] Table 2.2.4: Isolation of quercetin

[0145] Yield% Polyphenols% Quercetin% Experiment 1 2.8 93 91.4 Experiment 2 3.4 91.5 90.9 Experiment 3 3.3 92.3 91.8

[0146] Oleoresin from by-products of lutein esters

[0147] The by-product obtained by desolventizing the mother liquor showed similar qualities to conventional (hexane extracted) oleoresin as shown in Table 2.2.5. Saponification of the oleoresin gave free xanthophylls with a purity of >74%.

[0148] Table 2.2.5: Marigold oleoresin from lutein ester by-products

[0149]

[0150] Example 3: Extraction of lutein esters from freshly chopped marigold flowers using ethanol-acetone 3.1: Preparation of chopped marigold flowers for lutein ester extraction

[0151] Fresh marigold flowers, having a xanthophylls content of 0.04 to 0.3% by weight, a polyphenol and wax content of 8 to 11% by weight, and a moisture content of 88 to 92% by weight, are mixed with an optimized combination of enzymes, including cellulases, hemicellulases, cellulose biohydratases, pectinases, galactomannanases, proteases, and lipases. The enzyme mixture is dosed at a dosage range of 0.02 to 0.5% of the flower weight. The flower biomass is simultaneously ensiled and hydrolyzed for approximately 5 to 10 days to produce enzyme-ensiled biomass.

[0152] The ensiled biomass is dejuiced to remove surface moisture and then chopped to produce chopped marigold flowers. The chopped flowers have a xanthophylls content ranging from 0.5 to 1.2% and a xanthophyll content of 77 to 87%. The chopped marigold flowers are extracted with a polar solvent such as ethanol or methanol at a weight ratio of chopped marigold flowers to solvent ranging from 1:2 to 1:8, preferably 1:5. The mixture is preferably heated to a temperature ranging from 40 to 50°C with continuous solvent circulation for approximately 30 minutes. The extraction procedure is repeated to fully extract the water-soluble components and polyphenols from the flowers, thereby producing an ethanol fraction (polar solvent fraction) and residual biomass. The ethanol fraction is then filtered, and the ethanol is evaporated and then processed to isolate quercetin, the main flavonoid present in marigold flowers. The residual biomass is extracted with acetone at a weight ratio of residual biomass to acetone ranging from 1:2 to 1:8, preferably 1:4. The mixture is preferably heated to a temperature in the range of 40 to 45°C with continuous solvent circulation for approximately 30 minutes. The extraction procedure is repeated to completely extract the xanthophylls. The acetone fraction thus obtained is concentrated to obtain an extract having an extract content ranging from 1 to 5% extractables and maintained at 8 to 15°C for crystallization for 10 to 24 hours, preferably 12 hours. The crystallized lutein esters are filtered and dried in a dehumidified air dryer at 45 to 48°C. This produces high-purity lutein ester crystals ranging from 0.4 to 0.8% of the chopped marigold flower, wherein the purity of the trans-lutein ester in the isolated crystals is 78 to 85%, and the crystals further contain 2 to 5% trans-zeaxanthin esters. The mother liquor is concentrated to obtain an oleoresin with a yield of 0.8 to 1.6% relative to the chopped marigold flower, and a xanthophylls content of 12 to 16%. The trans-lutein content of this by-product (i.e., the oleoresin) is 68 to 74%.

[0153] 3.2: Laboratory-scale production of high-purity crystalline lutein esters from cut marigold flowers

[0154] 250 g of chopped marigold flowers were placed in a 2-L Erlenmeyer flask and treated with the specified amount of ethanol, as shown in Table 3.1. The mixture was heated at 40 to 50°C with stirring and extracted with the specified volume of ethanol. The mixture was then filtered, and the remaining biomass was re-extracted with ethanol until the ethanol-solubles were completely extracted, preferably for four extraction cycles. The remaining biomass was then treated with the specified amount of acetone, as shown in Table 3.1, at 40 to 45°C. Repeated extractions with acetone were performed to recover all xanthophylls from the biomass. The acetone oil mixture was concentrated to the specified concentration level, as shown in Table 3.1, and maintained for the specified time and temperature for crystallization. The settled crystals of trans-lutein esters were filtered and dried at 45 to 48°C under DHAD. Total carotenoids were estimated by UV-Vis spectroscopy, and xanthophylls were measured by HPLC after saponification.

[0155] Table 3.1: Experimental conditions for high purity lutein esters from cut marigold flowers

[0156]

[0157] 3.3: Large-scale production of high-purity crystalline lutein esters from chopped marigold flowers

[0158] 100 kg of chopped marigold flowers were placed in an extractor and treated with ethanol at a weight ratio of flowers to ethanol (1:5). The mixture was heated at 40 to 50°C with stirring and extracted with a specified volume of ethanol. The mixture was then filtered and the residue was re-extracted with ethanol until the ethanol-soluble matter was completely extracted, with four extraction cycles performed. The residual biomass was treated with acetone at a weight ratio of flowers to acetone (1:4) at 40 to 45°C. Repeated extractions with acetone were completed to recover all xanthophylls from the biomass. The acetone mixed oil was concentrated to a concentration of 2% and kept at 10°C for 12 hours for crystallization. The settled crystals of trans-lutein esters were filtered out and dried under DHAD at 45 to 48°C. Total carotenoids were estimated by UV-Vis and lutein esters were measured by HPLC after saponification.

[0159] Table 3.2: Product profile of high-purity lutein esters from chopped marigold flowers

[0160] experiment % Yield from Chopped Flowers % Total Carotenoids % Lutein Esters 1 0.64 87 81.8

[0161] Common impurities (pollutants) in marigold products are polycyclic aromatic hydrocarbons, dioxins, nitrosamines, ethylene oxide, etc.

[0162] Table 3.3: Impurity profile of high purity lutein esters from chopped marigold flowers

[0163] parameter result protein% 0.12 Ash % <0.1 PAHs (ug / kg) Not detected Benzopyrene (ug / kg) Not detected insecticides Not detected Dioxins, furans (pg / g) 0.488 Melamine (mg / kg) <0.1 Ethylene oxide (ppb) <10 Nitrosamines (mg / kg) <0.01

[0164] Ethanol extract (i.e. polar solvent fraction)

[0165] The ethanol oil mixture from the experiment was desolventized to produce an extract containing polyphenols as the active component. The main polyphenol in the extract was quercetin.

[0166] Table 3.4: Polyphenols from ethanol extracts

[0167] %Yield % polyphenols % Quercetin 1.8 34 24

[0168] Purification of the ethanol extract using carbon treatment in ethanol medium yielded quercetin with >90% purity.

[0169] Table 3.5: Isolation of quercetin from ethanol extract

[0170] %Yield % polyphenols % Quercetin Experiment 1 0.28 92.4 91.3

[0171] Oleoresin from by-products of lutein esters

[0172] The byproduct obtained by desolventizing the mother liquor exhibited qualities similar to conventional (hexane-extracted) oleoresin. Yields of the byproduct ranged from 0.8 to 1.6%, with 12 to 16% xanthophylls, including 68 to 74% trans-lutein, as shown in Table 3.6. The oleoresin could be saponified to yield free xanthophylls with a purity of >74%.

[0173] Table 3.6: Oleoresins from Lutein Esters

[0174] % oleoresin yield % Luteins % Lutein by HPLC % Zeaxanthin by HPLC 1.5 14 71 3.4

[0175] Example 4: Extraction of Lutein Esters from Marigold Powder Using Isopropyl Alcohol

[0176] Marigold powder (250 g) prepared using Example 1 was placed in a 5 L Erlenmeyer flask and treated with isopropanol in a weight ratio of powder to isopropanol ranging from 1:2 to 1:8, as shown in Table 4. The mixture was heated at 55°C with stirring and extracted with isopropanol for a period of 30 minutes. The mixture was then filtered and the residue was re-extracted with isopropanol until the isopropanol soluble content was completely extracted, preferably 4 extraction cycles. The isopropanol fraction was then concentrated to 5% to 10% of the extract to obtain an extract containing lutein esters, as shown in Table 4. The extract was then crystallized at 35°C for 24 to 72 hours, preferably 48 hours, as shown in Table 4. The crystals of trans-lutein ester that settled down were filtered out and dried under DHAD at 45 to 48°C.

[0177] Observations and results

[0178] The extraction method using isopropyl alcohol produced lutein ester crystals in the range of 2 to 3% of flour, wherein the purity of trans-lutein ester in the isolated crystals was 70 to 75%, and the crystals further contained 2 to 6% trans-zeaxanthin ester (Table 4). In particular, Experiment No. 2, which involved using a 1:5 flour:isopropyl alcohol weight ratio and crystallization at 35°C and 48 hours, produced 73.9% lutein ester crystals.

[0179] The mother liquor was concentrated to an oleoresin with a yield of 20 to 22% relative to the powder, and the xanthophylls content in the oleoresin was 5 to 8%. The trans-lutein content in this oleoresin was 65 to 70%. Specifically, the oleoresin, which contained 22.7% of the powder, contained 6.8% of xanthophylls. Table 4 shows working and non-working examples, with experiments 1 to 3 achieving satisfactory results. However, no crystals or oleoresin properties were observed in experiments 4, 5, and 6.

[0180] Table 4: Experimental conditions for high purity lutein esters extracted using isopropanol

[0181]

[0182] Advantages of the present disclosure

[0183] The present disclosure provides an efficient extraction method for obtaining an extract with high-purity crystalline lutein esters. Unlike conventional methods, the disclosed method does not involve hexane treatment of marigold powder / pellets, but directly uses marigold-flower / powder / pellets in an extraction procedure using environmentally friendly solvents (ethanol and acetone; or isopropanol). This is an innovative process with a smaller number of unit operations and a higher recovery rate of lutein in crystalline lutein esters. The disclosed method is a simple solvent extraction process and does not involve multi-stage laborious solvent purification procedures. Therefore, the method can be scaled up for the use of high-purity lutein ester crystals in pharmaceutical and dietary supplement compositions.

Claims

1. A method for preparing lutein esters, comprising: a) providing a raw material selected from chopped marigold flowers, marigold powder or a combination thereof; b) extracting the raw material with at least one polar solvent selected from ethanol, methanol or a combination thereof to obtain a polar solvent fraction and residual biomass; c) extracting the residual biomass with an extractant comprising acetone to obtain an acetone fraction; and d) concentrating the acetone fraction.

2. The process according to claim 1, wherein the concentration is carried out to obtain an extract comprising 1 to 12 wt% of extractives, preferably 1 to 5% of extractives when the raw material is chopped marigold flowers, preferably 5 to 10% of extractives when the raw material is marigold powder.

3. The method according to claim 2, further comprising crystallizing the extract to obtain crystals comprising lutein esters, and a mother liquor.

4. The method of claim 1, wherein the ratio of the raw material to the polar solvent is in the range of 1:2 to 1:

8.

5. The method according to claim 1, wherein step (b) is performed at a temperature ranging from 30 to 60°C with the solvent continuously circulated for a period of 20 to 100 minutes.

6. The method of claim 1, wherein the ratio of residual biomass to extractant is in the range of 1:2 to 1:

8.

7. The method of claim 1, wherein step (c) is performed at a temperature ranging from 25 to 52°C with continuous circulation of the extractant for 20 to 60 minutes.

8. The process according to claim 3, wherein the crystallization is carried out at a temperature in the range of 7 to 40°C for a period of 8 to 60 hours, preferably 10 to 30 hours.

9. The method according to claim 3, wherein the yield of the lutein ester is in the range of 0.25 to 2.5 wt% relative to the raw material, preferably 1.1 to 2.5 wt% when the raw material is marigold powder, or preferably 0.25 to 1.0 wt% when the raw material is chopped marigold flowers.

10. The process according to claim 3, wherein the purity of the lutein ester is at least 55 wt%, preferably at least 76 wt%, and wherein the crystals further comprise zeaxanthin ester in the range of 2 to 6 wt%.

11. The method of claim 3, further comprising concentrating the mother liquor to obtain an oleoresin, wherein the oleoresin comprises xanthophylls in the range of 12 to 16 wt %, of which 70 to 75 wt % are trans-lutein and 3 to 6 wt % are zeaxanthin.

12. The process according to claim 11, wherein the oleoresin is subjected to saponification to obtain free xanthophylls of at least 74% purity.

13. The method of claim 1, wherein the polar solvent fraction comprises at least one polyphenol selected from quercetin, 6-hydroxykaempferol, quercetin, tagetin, or a mixture thereof.

14. The method of claim 1, further comprising subjecting the polar solvent fraction to carbon purification in ethanol to obtain quercetin.

15. A method for preparing lutein esters, comprising: (a) providing a raw material selected from chopped marigold flowers, marigold powder or a combination thereof; (b) subjecting the raw material to extraction with an extractant comprising isopropanol to obtain an isopropanol fraction; and (c) concentrating the isopropanol fraction and optionally subsequently crystallizing to obtain lutein ester crystals.

16. The method according to claim 15, wherein the crystallization is performed to obtain crystals comprising lutein esters, and a mother liquor.

17. The method of claim 15, wherein the ratio of raw material to extractant is in the range of 1:2 to 1:8, preferably 1:

5.

18. The process according to claim 15, wherein step (b) is carried out at a temperature in the range of 50 to 60°C with continuous circulation of the solvent for a period of 20 to 40 minutes, preferably 30 minutes.

19. The method according to claim 15, wherein the crystallization is carried out at a temperature in the range of 10 to 55°C for a period of 5 to 90 hours.

20. The method according to claim 15, wherein the concentration is performed to obtain an extract comprising 1 to 12% extractables, preferably 5 to 12% extractables.

21. The method according to claim 15, wherein the yield of the lutein ester is in the range of 2 to 3 wt% relative to marigold powder.

22. The method of claim 15, wherein the purity of the lutein ester is at least 55 wt%, and wherein the crystals further comprise zeaxanthin ester in the range of 2 to 6 wt%.

23. The method of claim 16, further comprising concentrating the mother liquor to obtain an oleoresin, wherein the oleoresin comprises xanthophylls in the weight range of 5 to 8 wt %, wherein lutein is in the weight range of 70 to 75 wt % and zeaxanthin is in the weight range of 3 to 6 wt %.

24. The method according to claim 1, wherein the marigold powder is obtained by a process comprising: ensilaging marigold flowers for a period of preferably 5 to 10 days with at least one enzyme selected from the group consisting of cellulase, hemicellulase, cellulose biohydratase, pectinase, galactomannanase, protease, lipase and combinations thereof; and dehydrating to obtain the marigold powder.

25. The method according to any one of the preceding claims, wherein step (b) and / or step (c) is repeated at least once, preferably 2 to 5 times.

26. The method of claim 3, wherein the crystals contain protein in the weight range of 0 to 0.5%, ash in the weight range of 0 to 0.2%, polycyclic aromatic hydrocarbons (PAH) in an amount of 0 to 2 ppb, benzopyrene in an amount of 0 to 0.5 ppb, pesticides in an amount of 0 to 0.01 ppm, dioxins in an amount of 0 to 0.5 pg / g, melamine in an amount of 0 to 0.1 mg / kg, ethylene oxide in an amount of 0 to 10 ppb, and nitrosamines in an amount of 0 to 0.01 mg / kg.

27. The method of claim 14, wherein the quercetin is at least 90% pure.

28. The method according to claim 1, wherein the concentration is carried out to obtain an extract comprising xanthophylls in the weight range of 15 to 30 wt%, of which lutein is in the weight range of 70 to 80 wt% and zeaxanthin is in the weight range of 3 to 6 wt%.

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