WO2015067707A1 - Process for the purification of astaxanthin - Google Patents

Process for the purification of astaxanthin Download PDF

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Publication number
WO2015067707A1
WO2015067707A1 PCT/EP2014/073956 EP2014073956W WO2015067707A1 WO 2015067707 A1 WO2015067707 A1 WO 2015067707A1 EP 2014073956 W EP2014073956 W EP 2014073956W WO 2015067707 A1 WO2015067707 A1 WO 2015067707A1
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Prior art keywords
astaxanthin
synthetic
ppm
suspension
dichloromethane
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PCT/EP2014/073956
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French (fr)
Inventor
Philippe Karrer
Bettina Wuestenberg
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Dsm Ip Assets B.V.
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Publication of WO2015067707A1 publication Critical patent/WO2015067707A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
    • C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

  • the object of the invention is to provide a synthetic food-grade astaxanthin (AXN) which is not known so far.
  • Food-grade within the present invention means "suitable for human consumption”.
  • Such a food-grade synthetic AXN thus fulfills all regulatory requirements concerning purity and can be used in the commercial production of food and dietary supplements.
  • AXN can be manufactured by chemical synthesis (see EP-A 908 449, WO 2005/087720, EP-A 733 619), it can be produced by fermentation (see EP-A 543 023) and isolated from natural sources such as shell waste (see JP-A 11-049 972) or algae Haematococcus pluvialis (see GB-A 2,301,587), where the isolated esters would have to be cleaved to AXN itself.
  • AXN isolated from natural sources is already used in products for human consumption: see e.g.
  • Synthetic AXN is of standardized quality compared to AXN from natural sources since it is much easier to use a standardized procedure for its chemical synthesis and purification than being dependent from the varying quality of natural AXN resulting by using natural sources which vary also in their composition.
  • AXN means "synthetic AXN”.
  • Chemical processes for the manufacture of AXN are often carried out in halogenated hydrocarbons such as dichloromethane (see e.g. WO 2011/095571) since AXN and its precursors have a high solubility in these solvents though there were intentions in the past to avoid the use of such solvents (see e.g. EP-A 908 449 and WO 2005/087720).
  • chloroform see JP-A 07118226).
  • the thus obtained AXN is then often crystallized from lower alkanols since it is hardly soluble in such solvents.
  • the present invention is especially directed to a synthetic AXN with a content of dichloromethane ⁇ 250 ppm, preferably with a content of dichloromethane ⁇ 200 ppm, more preferably with a content of dichloromethane ⁇ 100 ppm, even more preferably with a content of dichloromethane ⁇ 50 ppm, ⁇ 35 ppm, ⁇ 30 ppm, ⁇ 25 ppm, ⁇ 20 ppm, most preferably with a content of dichloromethane ⁇ 10 ppm.
  • the synthetic AXN has a content of dichloromethane in the range of between 0 and 100 ppm, preferably in the range of between 10 and 100 ppm.
  • the synthetic AXN according to the present invention has a content of methanol ⁇ 500 ppm, preferably it has a content of methanol ⁇ 350 ppm, more preferably it has a content of methanol ⁇ 250 ppm, even more preferably it has a content of methanol ⁇ 150 ppm, ⁇ 100 ppm, most preferably it has a content of methanol ⁇ 50 ppm, ⁇ 20 ppm, ⁇ 10 ppm, ⁇ 5 ppm.
  • the synthetic AXN has a content of methanol in the range of between 0 and 50 ppm, preferably in the range of between 0 and 20 ppm, more preferably in the range of 0 to 10 ppm, most preferably in the range of 0 to 5 ppm.
  • the synthetic AXN has a content of dichloromethane ⁇ 50 ppm and a content of methanol ⁇ 100 ppm.
  • the AXN according to the present invention may especially be used in dietary supplements. For these purposes it is often provided in the form of an oily suspension or a powdery formulation such as a beadlet, thus protecting the synthetic AXN from degradation.
  • a further object of the present invention is a human dietary supplement comprising astaxanthin manufactured by chemical synthesis (i.e. synthetic astaxanthin), free from astaxanthin mono- and diesters, and comprising ⁇ 250 ppm of dichloromethane.
  • Preferred embodiments are human dietary supplements comprising this synthetic AXN, free from AXN mono- and diesters, comprising ⁇ 200 ppm of dichloromethane, ⁇ 100 ppm of dichloromethane, ⁇ 50 ppm of dichloromethane.
  • Further preferred embodiments are human dietary supplements comprising synthetic food-grade AXN, free from AXN mono- and diesters, comprising dichloromethane in the range between 0 and 100 ppm, preferably in the range between 10 and 100 ppm.
  • the present invention is directed to human dietary supplements comprising synthetic food-grade AXN, free from AXN mono-and diesters and with the reduced levels of dichloromethane and methanol as given above including all preferred ranges and combinations given. Since it is possible with the processes of the present invention to significantly reduce the content of halogenated organic solvents such as dichloromethane, there is no need any more to use a chemical synthesis, where the use of such solvents is avoided.
  • Such a synthetic food-grade AXN may be obtained by one of the following processes which are also objects of the present invention.
  • a process for the manufacture of synthetic food-grade astaxanthin comprising the following steps:
  • step b) Heating up the suspension obtained in step b) to a temperature in the range of from 110 to 150°C in a closed reactor;
  • step c) Maintaining the suspension at a temperature in the range as given for step c); and e) Optionally removing residual dichloromethane and ethyl acetate and, if present, methanol by distillation; and
  • step b) Repeating steps b), d) and e) until the content of dichloromethane in the synthetic astaxanthin is ⁇ 250 ppm, with the proviso that step b) is only repeated if step e) is performed or repeated;
  • step f) Cooling the synthetic astaxanthin suspension obtained in step f), whereby its content of dichloromethane is ⁇ 250 ppm, to a temperature in the range of from 10 to 35°C;
  • a process for the manufacture of synthetic food-grade astaxanthin comprising the following steps:
  • step bl Heating up the suspension obtained in step bl) to a temperature in the range of from 110 to 150°C in a closed reactor;
  • step dl Maintaining the suspension at a temperature in the range as given for step cl); and el) Removing residual dichloromethane, ethyl acetate and, if present, methanol by distillation; and
  • synthetic astaxanthin is ⁇ 250 ppm, with the proviso that step bl) is only repeated if step el) is performed or repeated;
  • step fl Cooling the synthetic astaxanthin suspension obtained in step fl), whereby its content of dichloromethane is ⁇ 250 ppm, to a temperature in the range of from 10 to 35°C;
  • synthetic AXN crystals As starting material either synthetic AXN crystals (see process A - step a)) or a solution of synthetic AXN in dichloromethane (see process B - step al)) may be used.
  • Process A The synthetic AXN crystals are as obtained from any of the chemical syntheses after removing of the solvent, in which the synthesis has been performed as e.g. preferably in dichloromethane.
  • the dichloromethane may have been removed by distillation or by solvent exchange.
  • the dichloromethane has been removed by a solvent exchange with methanol.
  • dichloromethane is heated up to a temperature of 38 to 40°C at atmospheric pressure and dichloromethane is distilled off. Simultaneously methanol is added so that the volume of the mixture is kept constant. Then solvent (dichloromethane + methanol) are distilled off until the internal temperature has been raised to 64°C (boiling point of methanol) meaning that only methanol is distilled off, but no dichloromethane any more.
  • dichloromethane in the solution by distillation Preferably this is done whereby simultaneously methanol is added.
  • steps a2) and a3) are also performed. a2) adding methanol to the synthetic astaxanthin solution of step al);
  • step b)/step bl) is carried out in a closed reactor.
  • the ethyl acetate is added to the synthetic astaxanthin crystals (step b) - Process A) or the solution of synthetic AXN in dichloromethane (step bl) - Process B) in an amount to obtain a suspension of synthetic astaxanthin in ethyl acetate, whereby the amount of synthetic astaxanthin in said suspension is in the range of from 20 to 60 weight-%, more preferably in the range of from 30 to 50 weight-%. Preferably this is done under nitrogen.
  • the suspension obtained in step b)/step bl) is heated up to a temperature, i.e. brought to a temperature, in the range of from 110 to 150°C, more preferably to a temperature in the range of from 110 to 140°C, even more preferably to a temperature in the range of from 110 to 125°C, in a closed reactor.
  • a temperature i.e. brought to a temperature, in the range of from 110 to 150°C, more preferably to a temperature in the range of from 110 to 140°C, even more preferably to a temperature in the range of from 110 to 125°C, in a closed reactor.
  • the temperature is maintained constant at the wanted value - the same temperature as in step c)/cl).
  • Step e) and step el), respectively, is optional, i.e. it can be performed or not. Preferably this step is performed.
  • methanol is also present or not, either dichloromethane, ethyl acetate and methanol or only dichloromethane and ethyl acetete are then removed by distillation.
  • dichloromethane and ethyl acetate and methanol, if present, are removed. This is performed until the content of dichloromethane in the synthetic astaxanthin is ⁇ 250 ppm, preferably ⁇ 200 ppm, more preferably ⁇ 100 ppm, even more preferably ⁇ 50 ppm, ⁇ 20 ppm, most preferably ⁇ 10 ppm.
  • steps b) feeding of ethyl acetate
  • step e removing of ethyl acetate and dichloromethane and, if present, methanol
  • steps bl) and el) are carried out simultaneously after the end temperature of step c) has been reached.
  • Process A and Process B respectively are performed as follows:
  • Process A Performing step a) and step b) and step d) and step g) and step h), meaning that the suspension of synthetic AXN in ethyl acetate is kept for a certain period of time at a temperature in the range of from 110 to 150°C until the content of dichloromethane in the synthetic AXN is ⁇ 250 ppm.
  • Process B Performing step al) and step bl) and step dl) and step gl) and step hi), meaning that the suspension of synthetic AXN in ethyl acetate is kept for a certain period of time at a temperature in the range of from 110 to 150°C until the content of dichloromethane in the synthetic AXN is ⁇ 250 ppm.
  • the synthetic astaxanthin suspension obtained in step f)/fl), whereby its content of dichloromethane is ⁇ 250 ppm, is cooled to a temperature in the range of from 15-30°C, more preferably to a temperature in the range of from 20 to 25°C.
  • the suspension obtained in step d)/dl) may be cooled down, if the removal of ethyl acetate and dichlormethane and methanol, if present, and the addition of fresh ethyl acetate is not repeated).
  • the synthetic astaxanthin crystals are then filtered off and optionally dried.
  • the drying of the obtained synthetic AXN crystals is generally carried out at a temperature below 140°C, preferably at a temperature in the range of from 40 to 140°C, and in vacuum.
  • the vacuum is preferably in a range of between 0 and 20 mbara.
  • the drying of the synthetic AXN crystals is carried out at a temperature of 80°C and at 20 mbar.
  • the drying of the synthetic AXN crystals is carried out at a temperature in the range of from 55 to 70°C and at a pressure below 20 mbar.
  • An embodiment of the present invention is a process, where the steps a) to h) and al) to hi), respectively, are carried out twice, meaning that the synthetic astaxanthin obtained in step h) or in step hi), respectively, is used as starting material for step a) and step al), respectively.
  • Especially preferred embodiments of the present invention are those processes, whereby one or more of the preferred conditions given above are realized.
  • the most preferred processes of the present invention are those, whereby all preferred conditions given above are realized.
  • the synthetic food-grade AXN of the present invention may be admixed with an edible oil to obtain an oily suspension of synthetic food-grade AXN.
  • the edible oil may be selected from the following group consisting of vegetable oils such as corn oil, safflower oil, sunflower oil, middle chain triglycerides (MCT) oil, peanut oil, soybean oil, rapeseed oil, palm oil, palm kernel oil, cotton seed oil, olive oil, coconut oil, and synthetic oils, and any mixture thereof, but is not limited thereto.
  • Especially preferred edible oils are selected from the group consisting of safflower oil, corn oil, sunflower oil, middle chain triglycerides (MCT) oil and any mixture thereof.
  • Such an oily suspension of synthetic food-grade AXN is also an object of the present invention.
  • the content of synthetic food-grade AXN in such a suspension is in the range of 0.5 to 20 weight-%, based on the total weight of the suspension.
  • the content of synthetic food-grade AXN in such a suspension is in the range of 1.0 to 10 weight-%, based on the total weight of the suspension.
  • the oily suspension may further comprise an antioxidant, especially a fat-soluble antioxidant.
  • An especially preferred fat-soluble antioxidant is dl-alpha-tocopherol.
  • the amount of such anti-oxidant is preferably in the range of 0.1 to 3 weight-%, based on the total weight of the suspension. The amount of the oil is then so much that the amount of all three ingredients (synthetic food-grade AXN; anti-oxidant, oil) sums up to 100 weight-%).
  • the oily suspension may be directly used as such for the manufacture of dietary supplements in the form capsules, especially in the form of soft-gel capsules.
  • the powdery forms may be used for making tablets.
  • An especially preferred powdery form is a beadlet comprising the synthetic food-grade astaxanthin as disclosed above encapsulated in a matrix comprising a modified food starch.
  • the amount of synthetic food-grade astaxanthin in said beadlet is preferably in the range of 0.5 to 20 weight-%, based on the total weight of the beadlet.
  • the beadlets may further comprise a water-soluble antioxidant (such as sodium ascorbate) and/or a fat-soluble antioxidant (such as DL-alpha-tocopherol), as well as a saccharide as adjuvant.
  • a water-soluble antioxidant such as sodium ascorbate
  • a fat-soluble antioxidant such as DL-alpha-tocopherol
  • saccharide as adjuvant.
  • the amount of each of the anti-oxidants is preferably in the range of 0.5 to 3.0 weight-%), based on the total weight of the beadlet.
  • a particularly preferred modified food starch of this invention has the following formula (I)
  • R is an alkylene radical and R ' is a hydrophobic group.
  • R is a lower alkylene radical such as dimethylene or trimethylene.
  • R ' may be an alkyl or alkenyl group, preferably having 5 to 18 carbon atoms.
  • a preferred compound of formula (I) is an "OSA-starch” (starch sodium octenyl succinate).
  • the degree of substitution i.e. the number of esterified hydroxyl groups to the number of free non-esterified hydroxyl groups usually varies in a range of from 0.1% to 10%, preferably in a range of from 0.5% to 4%, more preferably in a range of from 3% to 4%.
  • OSA-starch denotes any starch (from any natural source such as corn, waxy maize, waxy corn, wheat, tapioca and potato or synthesized) that was treated with octenyl succinic anhydride (OSA).
  • the degree of substitution i.e. the number of hydroxyl groups esterified with OSA to the number of free non-esterified hydroxyl groups usually varies in a range of from 0.1% to 10%, preferably in a range of from 0.5% to 4%, more preferably in a range of from 3% to 4%.
  • OSA-starches are also known under the expression "modified food starch".
  • OSA-starches encompasses also such starches that are commercially available e.g. from National Starch under the tradenames HiCap 100, Capsul
  • a commercially available modified food starch such as e.g. HiCap 100 (from National Starch) and ClearGum Co03 (from Roquette Freres) is used. It is especially advantageous if such a starch or an OSA starch in general is further improved according to a process as disclosed in WO 2007/090614, especially according to a procedure as described in examples 28, 35 and/or 36 of WO 2007/090614.
  • such a commercially available starch has been centrifuged as an aqueous solution or suspension before use.
  • the centrifugation may be carried out at 1000 to 20000 g depending on the dry mass content of the modified polysaccharide in the aqueous solution or suspension. If the dry mass content of the modified polysaccharide in the aqueous solution or suspension is high, the applied centrifugation force is also high. For example for an aqueous solution or suspension with a dry mass content of the modified polysaccharide of 30 weight-% a centrifugation force of 12000 g may be suitable to achieve the desired separation.
  • the centrifugation may be carried out at dry matter contents in the range of from 0.1-60 weight %, preferably in the range of from 10-50 weight-%, most preferably in the range of from 15-40 weight-% at temperatures in the range of from 2-99°C, preferably in the range of from 10-75°C, most preferably in the range of from 40-60°C.
  • Preferred monosaccharides are glucose and fructose, as well as any mixture thereof.
  • glucose in the context of the present invention does not only mean the pure substance, but also a glucose syrup with a DE > 90. This also applies for the other monosaccharides.
  • Dextrose equivalent denotes the degree of hydrolysis and is a measure of the amount of reducing sugar calculated as D-glucose based on dry weight; the scale is based on native starch having a DE close to 0 and glucose having a DE of 100.
  • disaccharides are saccharose, isomaltose, lactose, maltose and nigerose, as well as any mixture thereof.
  • An example of an oligosaccharide is maltodextrin.
  • An example of a polysaccharide is dextrin.
  • invert sugar glucose + fructose + saccharose
  • Glucidex IT 47 from Roquette Freres
  • Dextrose Monohydrate ST from Roquette Freres
  • Sirodex 331 from Tate & Lyle
  • Glucamyl F 452 from Tate & Lyle
  • the amount of the modified food starch is in the range of from 20 to 80 weight-%, more preferably in the range of from 40 to 60 weight-%, and the amount of the saccharide (adjuvant) is in the range of from 5 to 30 weight-%, based on the total amount of the composition.
  • the beadlets are manufactured according to the process described below, whereby especially corn starch is used as powder-catch agent. Most preferably the thus resulting beadlets contain from 10 to 25 weight-% of corn starch, based on the total weight of the beadlet.
  • the present invention is also directed to a human dietary supplement comprising synthetic food-grade AXN, free from AXN mono- and diesters, and comprising ⁇ 250 ppm of dichloromethane.
  • Such human dietary supplement preferably comprises ⁇ 500 ppm of methanol.
  • any synthetic food-grade AXN as described above (with all the preferences given) may be used.
  • human dietary supplements in the context of the present invention mean dietary supplements to be administered to and to be consumed by humans.
  • a further embodiment of the present invention is a method of marketing the synthetic food-grade AXN according to the present invention, as well as a method of marketing the dietary supplements for human consumption as mentioned above.
  • a preferred embodiment is a method of marketing synthetic food-grade astaxanthin comprising providing a kit said kit comprising i) synthetic food-grade astaxanthin according to the present invention with all preferences as given above;
  • a further preferred embodiment is a method of marketing a human dietary supplement comprising synthetic food-grade AXN, free from AXN mono- and diesters, and comprising ⁇ 250 ppm of dichloromethane, comprising providing a kit said kit comprising
  • AXN mono- and diesters comprising ⁇ 250 ppm of dichloromethane according to the present invention with all preferences as given above;
  • Such optional further information may be information about the dosage of such synthetic food-grade astaxanthin and such human dietary supplement, respectively, or any other useful information with respect to the administration to humans.
  • the invention is now further illustrated in the following non-limiting examples.
  • Example 5 Example 5
  • the crystals are washed with 2 x 190 ml of methanol and dried in a drying oven 16 hours at 80°C under 20 mbara. 115.1 g of dry astaxanthin are obtained containing 48 mg/kg of dichloromethane.
  • Example 5 is repeated, but instead of 7 hours of digestion time, 16 hours of digestion time are applied. 115.1 g of dry astaxanthin are obtained containing 38 mg/kg of dichloromethane.
  • Example 8 is repeated, but instead of 4 hours of digestion time, 7.5 hours of digestion time are applied. Furthermore, the distillate is removed and fresh ethyl acetate are added with a flow rate of 2.7 ml per minute. 118.9 g of dry astaxanthin are obtained containing 80 mg/kg of dichloromethane.
  • Example 13 Astaxanthin 10% Fluid Suspension (an oily suspension according to the present invention)
  • a coarse suspension which contains 60 g of purified crystalline astaxanthin as obtained in example 5, 5.5 g of dl-a-tocopherol and 480 g of safflower oil is prepared under mixing at 45°C.
  • this coarse suspension is pumped continuously through an agitated ball mill (diameter of the zirconiumdioxide pearls: 0.4 mm,
  • the first digestion was carried out in analogy to the above procedure in ethyl acetate (example 1) with some minor changes: 25.0 g (97.9 wt%) of astaxanthin containing 2214 mg/kg of dichloromethane and 197 mg/kg of methanol, 130 ml of ethyl acetate.
  • the oil bath was removed and the suspension was allowed to cool to 40°C for 1 hour (without active cooling) and to 25°C for 1 hour with waterbath-cooling.
  • the filter cake was dried: 16 hours at 60°C (10 mbar). After drying 22.86 g of dark violet astaxanthin crystals were obtained.

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Abstract

The present invention is directed to synthetic astaxanthin which is suitable for human consumption. The present invention is further directed to processes where ethyl acetate is used to drastically reduce the content of dichloromethane and methanol, if present, in such synthetic astaxanthin. The present invention is further directed to suspensions and beadlets comprising such synthetic food-grade astaxanthin and to human dietary supplements comprising this synthetic food-grade AXN, free from AXN mono- and diesters, comprising ≤ 250 ppm of dichloromethane and preferably comprising ≤ 500 ppm of methanol.

Description

Process for the purification of astaxanthin
The object of the invention is to provide a synthetic food-grade astaxanthin (AXN) which is not known so far. "Food-grade" within the present invention means "suitable for human consumption". Such a food-grade synthetic AXN thus fulfills all regulatory requirements concerning purity and can be used in the commercial production of food and dietary supplements.
AXN can be manufactured by chemical synthesis (see EP-A 908 449, WO 2005/087720, EP-A 733 619), it can be produced by fermentation (see EP-A 543 023) and isolated from natural sources such as shell waste (see JP-A 11-049 972) or algae Haematococcus pluvialis (see GB-A 2,301,587), where the isolated esters would have to be cleaved to AXN itself. AXN isolated from natural sources is already used in products for human consumption: see e.g. US 2009/0297492, where AXN is described as improving the cognitive performance; US 2009/0018210, where AXN is described as promoting fat degradation; EP-A 1 867 327, where AXN is described for preventing a neurodegenerative disease - to name only a few. So far, however, synthetic AXN was not used in products for human consumption.
Synthetic AXN is of standardized quality compared to AXN from natural sources since it is much easier to use a standardized procedure for its chemical synthesis and purification than being dependent from the varying quality of natural AXN resulting by using natural sources which vary also in their composition. Thus, in the context of the present invention the term "AXN" means "synthetic AXN". Chemical processes for the manufacture of AXN are often carried out in halogenated hydrocarbons such as dichloromethane (see e.g. WO 2011/095571) since AXN and its precursors have a high solubility in these solvents though there were intentions in the past to avoid the use of such solvents (see e.g. EP-A 908 449 and WO 2005/087720). There are also purification methods that use chloroform (see JP-A 07118226). The thus obtained AXN is then often crystallized from lower alkanols since it is hardly soluble in such solvents.
It was therefore an object of the present invention to provide a synthetic AXN which is of reliable quality, easy to obtain also in an industrial scale and suitable for human consumption.
Thus, it was an object of the present invention to provide a food-grade synthetic AXN. This object is met by the process of the present invention which yields an AXN with extremely low solvent levels, preferably with extremely low levels of dichloromethane, thus rendering this AXN suitable for human consumption. Such an AXN is also an object of the present invention.
Thus, the present invention is especially directed to a synthetic AXN with a content of dichloromethane < 250 ppm, preferably with a content of dichloromethane < 200 ppm, more preferably with a content of dichloromethane < 100 ppm, even more preferably with a content of dichloromethane < 50 ppm, < 35 ppm, < 30 ppm, < 25 ppm, < 20 ppm, most preferably with a content of dichloromethane < 10 ppm.
In an especially preferred embodiment of the present invention the synthetic AXN has a content of dichloromethane in the range of between 0 and 100 ppm, preferably in the range of between 10 and 100 ppm.
Preferably the synthetic AXN according to the present invention has a content of methanol < 500 ppm, preferably it has a content of methanol < 350 ppm, more preferably it has a content of methanol < 250 ppm, even more preferably it has a content of methanol < 150 ppm, < 100 ppm, most preferably it has a content of methanol < 50 ppm, < 20 ppm, < 10 ppm, < 5 ppm.
In an especially preferred embodiment of the present invention the synthetic AXN has a content of methanol in the range of between 0 and 50 ppm, preferably in the range of between 0 and 20 ppm, more preferably in the range of 0 to 10 ppm, most preferably in the range of 0 to 5 ppm.
In a further preferred embodiment the synthetic AXN has a content of dichloromethane < 50 ppm and a content of methanol < 100 ppm.
The AXN according to the present invention may especially be used in dietary supplements. For these purposes it is often provided in the form of an oily suspension or a powdery formulation such as a beadlet, thus protecting the synthetic AXN from degradation.
Thus, a further object of the present invention is a human dietary supplement comprising astaxanthin manufactured by chemical synthesis (i.e. synthetic astaxanthin), free from astaxanthin mono- and diesters, and comprising < 250 ppm of dichloromethane. Preferred embodiments are human dietary supplements comprising this synthetic AXN, free from AXN mono- and diesters, comprising < 200 ppm of dichloromethane, < 100 ppm of dichloromethane, < 50 ppm of dichloromethane. Further preferred embodiments are human dietary supplements comprising synthetic food-grade AXN, free from AXN mono- and diesters, comprising dichloromethane in the range between 0 and 100 ppm, preferably in the range between 10 and 100 ppm.
Moreover, the present invention is directed to human dietary supplements comprising synthetic food-grade AXN, free from AXN mono-and diesters and with the reduced levels of dichloromethane and methanol as given above including all preferred ranges and combinations given. Since it is possible with the processes of the present invention to significantly reduce the content of halogenated organic solvents such as dichloromethane, there is no need any more to use a chemical synthesis, where the use of such solvents is avoided.
Processes
Such a synthetic food-grade AXN may be obtained by one of the following processes which are also objects of the present invention.
Process A
A process for the manufacture of synthetic food-grade astaxanthin comprising the following steps:
a) Providing synthetic astaxanthin crystals containing dichloromethane and
optionally methanol; and
b) Adding ethyl acetate to the synthetic astaxanthin crystals in an amount to obtain a suspension of synthetic astaxanthin in ethyl acetate, whereby the amount of synthetic astaxanthin in said suspension is in the range of from 5 to 70 weight-%, based on the total weight of the suspension; and
c) Heating up the suspension obtained in step b) to a temperature in the range of from 110 to 150°C in a closed reactor; and
d) Maintaining the suspension at a temperature in the range as given for step c); and e) Optionally removing residual dichloromethane and ethyl acetate and, if present, methanol by distillation; and
f) Repeating steps b), d) and e) until the content of dichloromethane in the synthetic astaxanthin is < 250 ppm, with the proviso that step b) is only repeated if step e) is performed or repeated; and
g) Cooling the synthetic astaxanthin suspension obtained in step f), whereby its content of dichloromethane is < 250 ppm, to a temperature in the range of from 10 to 35°C; and
h) Filtering off the synthetic astaxanthin crystals and optionally drying them; whereby a synthetic food-grade astaxanthin suitable for human consumption is obtained.
Process B
A process for the manufacture of synthetic food-grade astaxanthin comprising the following steps:
al) Providing a solution of synthetic astaxanthin in dichloromethane; this solution optionally comprising methanol; and
a2) optionally adding methanol to the synthetic astaxanthin solution of step al); and a3) removing dichloromethane and optionally methanol by distillation; and bl) Adding ethyl acetate to the synthetic astaxanthin solution in an amount to obtain a suspension of synthetic astaxanthin, whereby the amount of synthetic astaxanthin in said suspension is in the range of from 5 to 70 weight-%, based on the total weight of the suspension; and
cl) Heating up the suspension obtained in step bl) to a temperature in the range of from 110 to 150°C in a closed reactor; and
dl) Maintaining the suspension at a temperature in the range as given for step cl); and el) Removing residual dichloromethane, ethyl acetate and, if present, methanol by distillation; and
fl) Repeating steps bl), dl) and el) until the content of dichloromethane in the
synthetic astaxanthin is < 250 ppm, with the proviso that step bl) is only repeated if step el) is performed or repeated; and
gl) Cooling the synthetic astaxanthin suspension obtained in step fl), whereby its content of dichloromethane is < 250 ppm, to a temperature in the range of from 10 to 35°C; and
hi) Filtering off the synthetic astaxanthin crystals and optionally drying them;
whereby a synthetic food-grade astaxanthin suitable for human consumption is obtained. These processes will now be described in more detail below. They are economic and may be performed in an industrial scale. During the processes according to the present invention the total amount of astaxanthin is not completely dissolved in the ethyl acetate. This is in contrast to a crystallization process where the total amount of astaxanthin is completely dissolved upon heating and re-crystallizes when the temperature is decreased again.
Since the steps of Process A and B only differ with respect to the starting material, they are the same and thus, described together.
Step aVstep al)
As starting material either synthetic AXN crystals (see process A - step a)) or a solution of synthetic AXN in dichloromethane (see process B - step al)) may be used. Process A: The synthetic AXN crystals are as obtained from any of the chemical syntheses after removing of the solvent, in which the synthesis has been performed as e.g. preferably in dichloromethane. The dichloromethane may have been removed by distillation or by solvent exchange. Preferably the dichloromethane has been removed by a solvent exchange with methanol. In this case the mixture of astaxanthin and
dichloromethane is heated up to a temperature of 38 to 40°C at atmospheric pressure and dichloromethane is distilled off. Simultaneously methanol is added so that the volume of the mixture is kept constant. Then solvent (dichloromethane + methanol) are distilled off until the internal temperature has been raised to 64°C (boiling point of methanol) meaning that only methanol is distilled off, but no dichloromethane any more.
These synthetic AXN crystals still contain dichloromethane which cannot be removed by just prolonging the time for drying these synthetic AXN crystals, even if the temperature and/or the vacuum is/are increased. Typical amounts of remaining dichloromethane in the AXN crystals are 0.2 to 0.3 weight-%, based on the total weight of the synthetic AXN crystals. Furthermore, these AXN crystals may also contain small amounts of methanol. Process B: Since the process of the present invention is very effective in removing dichloromethane, it is also possible to use directly a solution of the synthetic AXN in dichloromethane. In this case it is advantageous to first reduce the amount of
dichloromethane in the solution by distillation. Preferably this is done whereby simultaneously methanol is added.
Thus, if process B is performed advantageously steps a2) and a3) are also performed. a2) adding methanol to the synthetic astaxanthin solution of step al);
a3) removing dichloromethane and methanol by distillation.
Furthermore also a solution of synthetic astaxanthin in dichloromethane can be used which also contains methanol since the methanol is also effectively removed by the process of the present invention. Depending on the methanol content of such a solution it may not be necessary to add additional methanol in step a2).
Step bVstep bl
Preferably step b)/step bl) is carried out in a closed reactor.
Preferably the ethyl acetate is added to the synthetic astaxanthin crystals (step b) - Process A) or the solution of synthetic AXN in dichloromethane (step bl) - Process B) in an amount to obtain a suspension of synthetic astaxanthin in ethyl acetate, whereby the amount of synthetic astaxanthin in said suspension is in the range of from 20 to 60 weight-%, more preferably in the range of from 30 to 50 weight-%. Preferably this is done under nitrogen. Step cVStep cl)
Preferably the suspension obtained in step b)/step bl) is heated up to a temperature, i.e. brought to a temperature, in the range of from 110 to 150°C, more preferably to a temperature in the range of from 110 to 140°C, even more preferably to a temperature in the range of from 110 to 125°C, in a closed reactor. Thereby the pressure increases.
Step dVstep dl)
The temperature is maintained constant at the wanted value - the same temperature as in step c)/cl).
Step eVstep el)
Step e) and step el), respectively, is optional, i.e. it can be performed or not. Preferably this step is performed. Depending on whether methanol is also present or not, either dichloromethane, ethyl acetate and methanol or only dichloromethane and ethyl acetete are then removed by distillation.
Figure imgf000009_0001
If either dichloromethane, ethyl acetate and methanol or only dichloromethane and ethyl acetete are removed by distillation (= step e)/el)), step b)/bl), i.e. the addition of ethyl acetate, is repeated. Thus, the addition of fresh ethyl acetate is carried out and
simultaneously dichloromethane and ethyl acetate and methanol, if present, are removed. This is performed until the content of dichloromethane in the synthetic astaxanthin is < 250 ppm, preferably < 200 ppm, more preferably < 100 ppm, even more preferably < 50 ppm, < 20 ppm, most preferably < 10 ppm.
Thus, in a preferred embodiment of the present invention steps b) (feeding of ethyl acetate) and step e) (removing of ethyl acetate and dichloromethane and, if present, methanol) (process A) and steps bl) and el) (process B), respectively, are carried out simultaneously after the end temperature of step c) has been reached.
Alternatively Process A and Process B, respectively are performed as follows:
Process A: Performing step a) and step b) and step d) and step g) and step h), meaning that the suspension of synthetic AXN in ethyl acetate is kept for a certain period of time at a temperature in the range of from 110 to 150°C until the content of dichloromethane in the synthetic AXN is < 250 ppm. Process B: Performing step al) and step bl) and step dl) and step gl) and step hi), meaning that the suspension of synthetic AXN in ethyl acetate is kept for a certain period of time at a temperature in the range of from 110 to 150°C until the content of dichloromethane in the synthetic AXN is < 250 ppm.
Step gVstep l)
Preferably the synthetic astaxanthin suspension obtained in step f)/fl), whereby its content of dichloromethane is < 250 ppm, is cooled to a temperature in the range of from 15-30°C, more preferably to a temperature in the range of from 20 to 25°C.
Instead of the suspension obtained in step f)/fl) the suspension obtained in step d)/dl) may be cooled down, if the removal of ethyl acetate and dichlormethane and methanol, if present, and the addition of fresh ethyl acetate is not repeated). Step hVstep hi)
The synthetic astaxanthin crystals are then filtered off and optionally dried.
The drying of the obtained synthetic AXN crystals is generally carried out at a temperature below 140°C, preferably at a temperature in the range of from 40 to 140°C, and in vacuum. The vacuum is preferably in a range of between 0 and 20 mbara. In one embodiment of the present invention the drying of the synthetic AXN crystals is carried out at a temperature of 80°C and at 20 mbar. In a further embodiment of the present invention the drying of the synthetic AXN crystals is carried out at a temperature in the range of from 55 to 70°C and at a pressure below 20 mbar.
An embodiment of the present invention is a process, where the steps a) to h) and al) to hi), respectively, are carried out twice, meaning that the synthetic astaxanthin obtained in step h) or in step hi), respectively, is used as starting material for step a) and step al), respectively.
Especially preferred embodiments of the present invention are those processes, whereby one or more of the preferred conditions given above are realized. The most preferred processes of the present invention are those, whereby all preferred conditions given above are realized.
Oily suspensions
The synthetic food-grade AXN of the present invention may be admixed with an edible oil to obtain an oily suspension of synthetic food-grade AXN. The edible oil may be selected from the following group consisting of vegetable oils such as corn oil, safflower oil, sunflower oil, middle chain triglycerides (MCT) oil, peanut oil, soybean oil, rapeseed oil, palm oil, palm kernel oil, cotton seed oil, olive oil, coconut oil, and synthetic oils, and any mixture thereof, but is not limited thereto. Especially preferred edible oils are selected from the group consisting of safflower oil, corn oil, sunflower oil, middle chain triglycerides (MCT) oil and any mixture thereof.
Such an oily suspension of synthetic food-grade AXN is also an object of the present invention. Generally the content of synthetic food-grade AXN in such a suspension is in the range of 0.5 to 20 weight-%, based on the total weight of the suspension. Preferably the content of synthetic food-grade AXN in such a suspension is in the range of 1.0 to 10 weight-%, based on the total weight of the suspension. More preferably oily suspensions with a content of synthetic food-grade AXN of 5 and 10 weight- %, respectively, are marketed.
The oily suspension may further comprise an antioxidant, especially a fat-soluble antioxidant. An especially preferred fat-soluble antioxidant is dl-alpha-tocopherol. The amount of such anti-oxidant is preferably in the range of 0.1 to 3 weight-%, based on the total weight of the suspension. The amount of the oil is then so much that the amount of all three ingredients (synthetic food-grade AXN; anti-oxidant, oil) sums up to 100 weight-%).
The oily suspension may be directly used as such for the manufacture of dietary supplements in the form capsules, especially in the form of soft-gel capsules.
Powdery forms
The powdery forms may be used for making tablets. An especially preferred powdery form is a beadlet comprising the synthetic food-grade astaxanthin as disclosed above encapsulated in a matrix comprising a modified food starch.
The amount of synthetic food-grade astaxanthin in said beadlet is preferably in the range of 0.5 to 20 weight-%, based on the total weight of the beadlet.
The beadlets may further comprise a water-soluble antioxidant (such as sodium ascorbate) and/or a fat-soluble antioxidant (such as DL-alpha-tocopherol), as well as a saccharide as adjuvant. The amount of each of the anti-oxidants is preferably in the range of 0.5 to 3.0 weight-%), based on the total weight of the beadlet. A particularly preferred modified food starch of this invention has the following formula (I)
Figure imgf000013_0001
wherein St is a starch, R is an alkylene radical and R' is a hydrophobic group. Preferably R is a lower alkylene radical such as dimethylene or trimethylene. R' may be an alkyl or alkenyl group, preferably having 5 to 18 carbon atoms. A preferred compound of formula (I) is an "OSA-starch" (starch sodium octenyl succinate). The degree of substitution, i.e. the number of esterified hydroxyl groups to the number of free non-esterified hydroxyl groups usually varies in a range of from 0.1% to 10%, preferably in a range of from 0.5% to 4%, more preferably in a range of from 3% to 4%. The term "OSA-starch" denotes any starch (from any natural source such as corn, waxy maize, waxy corn, wheat, tapioca and potato or synthesized) that was treated with octenyl succinic anhydride (OSA). The degree of substitution, i.e. the number of hydroxyl groups esterified with OSA to the number of free non-esterified hydroxyl groups usually varies in a range of from 0.1% to 10%, preferably in a range of from 0.5% to 4%, more preferably in a range of from 3% to 4%. OSA-starches are also known under the expression "modified food starch".
The term "OSA-starches" encompasses also such starches that are commercially available e.g. from National Starch under the tradenames HiCap 100, Capsul
(octenylbutanedioate amy lo dextrin), Capsul HS, Purity Gum 2000, Clear Gum Co03,
UNI-PURE, HYLON VII; from National Starch and Roquette Freres, respectively; from CereStar under the tradename C*EmCap or from Tate & Lyle. In a preferred embodiment of the present invention a commercially available modified food starch such as e.g. HiCap 100 (from National Starch) and ClearGum Co03 (from Roquette Freres) is used. It is especially advantageous if such a starch or an OSA starch in general is further improved according to a process as disclosed in WO 2007/090614, especially according to a procedure as described in examples 28, 35 and/or 36 of WO 2007/090614.
Thus, in a further improved embodiment of the present invention such a commercially available starch has been centrifuged as an aqueous solution or suspension before use. The centrifugation may be carried out at 1000 to 20000 g depending on the dry mass content of the modified polysaccharide in the aqueous solution or suspension. If the dry mass content of the modified polysaccharide in the aqueous solution or suspension is high, the applied centrifugation force is also high. For example for an aqueous solution or suspension with a dry mass content of the modified polysaccharide of 30 weight-% a centrifugation force of 12000 g may be suitable to achieve the desired separation.
The centrifugation may be carried out at dry matter contents in the range of from 0.1-60 weight %, preferably in the range of from 10-50 weight-%, most preferably in the range of from 15-40 weight-% at temperatures in the range of from 2-99°C, preferably in the range of from 10-75°C, most preferably in the range of from 40-60°C.
The term„saccharide" in the context of the present invention encompasses mono-, di-, oligo- and polysaccharides, as well as any mixtures thereof.
Examples of monosaccharides are fructose, glucose (= dextrose), mannose, galactose, sorbose, as well as any mixtures thereof.
Preferred monosaccharides are glucose and fructose, as well as any mixture thereof. The term "glucose" in the context of the present invention does not only mean the pure substance, but also a glucose syrup with a DE > 90. This also applies for the other monosaccharides. The term "dextrose equivalent" (DE) denotes the degree of hydrolysis and is a measure of the amount of reducing sugar calculated as D-glucose based on dry weight; the scale is based on native starch having a DE close to 0 and glucose having a DE of 100.
Examples of disaccharides are saccharose, isomaltose, lactose, maltose and nigerose, as well as any mixture thereof.
An example of an oligosaccharide is maltodextrin.
An example of a polysaccharide is dextrin.
An example of a mixture of mono- and disaccharides is invert sugar (glucose + fructose + saccharose).
Mixtures of mono- and polysaccharides are e.g. commercially available under the tradenames Glucidex IT 47 (from Roquette Freres), Dextrose Monohydrate ST (from Roquette Freres), Sirodex 331 (from Tate & Lyle) and Glucamyl F 452 (from Tate & Lyle).
In a preferred embodiment the amount of the modified food starch is in the range of from 20 to 80 weight-%, more preferably in the range of from 40 to 60 weight-%, and the amount of the saccharide (adjuvant) is in the range of from 5 to 30 weight-%, based on the total amount of the composition.
More preferably the beadlets are manufactured according to the process described below, whereby especially corn starch is used as powder-catch agent. Most preferably the thus resulting beadlets contain from 10 to 25 weight-% of corn starch, based on the total weight of the beadlet.
Preferred are beadlets whose preparation comprises the following steps:
a) providing a modified food starch, a saccharide, a water-/fat- soluble antioxidant and a synthetic food-grade AXN according to the present invention;
b) preparing an aqueous nano-emulsion of said ingredients;
c) converting the nano-emulsion into a beadlet by a powder-catch process.
Human dietary supplements
The present invention is also directed to a human dietary supplement comprising synthetic food-grade AXN, free from AXN mono- and diesters, and comprising < 250 ppm of dichloromethane.
Such human dietary supplement preferably comprises < 500 ppm of methanol.
For these human dietary supplements any synthetic food-grade AXN as described above (with all the preferences given) may be used.
"human dietary supplements" in the context of the present invention mean dietary supplements to be administered to and to be consumed by humans.
A further embodiment of the present invention is a method of marketing the synthetic food-grade AXN according to the present invention, as well as a method of marketing the dietary supplements for human consumption as mentioned above.
A preferred embodiment is a method of marketing synthetic food-grade astaxanthin comprising providing a kit said kit comprising i) synthetic food-grade astaxanthin according to the present invention with all preferences as given above;
ii) information describing the benefits of said synthetic food-grade astaxanthin; iii) optional further information.
A further preferred embodiment is a method of marketing a human dietary supplement comprising synthetic food-grade AXN, free from AXN mono- and diesters, and comprising < 250 ppm of dichloromethane, comprising providing a kit said kit comprising
i) a human dietary supplement comprising synthetic food-grade AXN, free from
AXN mono- and diesters, and comprising < 250 ppm of dichloromethane according to the present invention with all preferences as given above;
ii) information describing the benefits of said human dietary supplement;
iii) optional further information.
Such optional further information may be information about the dosage of such synthetic food-grade astaxanthin and such human dietary supplement, respectively, or any other useful information with respect to the administration to humans. The invention is now further illustrated in the following non-limiting examples.
Examples
Examples 1-10: Processes according to the present invention
Example 1
In a 250 ml autoclave equipped with stirrer and thermometer, 50.0 g (97.9 wt%) of astaxanthin containing 2214 mg/kg of dichloromethane and 197 mg/kg of methanol were suspended in 130 ml of ethyl acetate. The autoclave was flushed with argon (3 minutes) and sealed. Stirring was started and adjusted to 570 rpm. The oil bath was heated to 140°C. The suspension was stirred for 16 hours at Tinternai = 123°C, whereby an over pressure of 2.7 barg was built up. After that, the oil bath was removed. The suspension inside the autoclave was allowed to cool for 1 hour at room temperature and was then cooled to Tinternai = 23°C. The autoclave was opened. The suspension was filtered and the filter cake was washed with methanol (2 x 25 ml). The resulting crystalline astaxanthin was first dried for 4 hours (60°C, 10 mbar) and then for another 16 hours (120°C, 3 x 10"2 mbar). After drying, dark violet astaxanthin crystals (yield: 95.4%) were obtained and subjected to GC head space and HPLC analysis: content of dichloromethane = 72 ppm; content of methanol = 0 ppm; content of ethyl acetate = 1570 ppm.
Example 2
Example 2 was carried out in analogy to example 1 with some changes: 15.0 g (97.9 wt%) of astaxanthin, 130 ml of ethyl acetate. Ti = 116°C, p = 2.7 barg. Results: 12.71 g of dark violet astaxanthin crystals; yield = 85%; content of dichloromethane = 108 ppm; content of methanol = 2 ppm; content of ethyl acetate = 1406 ppm.
Example 3
Example 3 was carried out in analogy to example 1 with some changes: 25.0 g (97.9 wt%) of astaxanthin, 130 ml of ethyl acetate. Ti = 119°C, p = 2.9 barg. Results: yield = 90.2%; content of dichloromethane = 86 ppm; content of methanol = 2 ppm; content of ethyl acetate = 1456 ppm.
Example 4
Example 4 was carried out in analogy to example 1 with some changes: 35.0 g (97.9 wt%) of astaxanthin, 130 ml of ethyl acetate. Ti = 123°C, p = 2.9 barg. Results: 33.42 g of dark violet astaxanthin crystals; yield = 97.0%; content of dichloromethane = 66 ppm; content of methanol = 1 ppm; content of ethyl acetate = 1529 ppm. Example 5
An autoclave is inertized with nitrogen, 120 g of AXN and 280 ml of ethyl acetate are introduced into the autoclave, the autoclave is re -inertized with nitrogen and closed, stirring is started (350 rpm), the internal temperature is set up to 120-121°C (reached in ~ 30 minutes) and kept constant during 7 hours of digestion time (internal pressure = 3.5 barg). Then the internal temperature is reduced to 20-25°C in ~ 30 minutes, the autoclave is opened and the slurry is filtered by a Buchner filter.
The crystals are washed with 2 x 190 ml of methanol and dried in a drying oven 16 hours at 80°C under 20 mbara. 115.1 g of dry astaxanthin are obtained containing 48 mg/kg of dichloromethane.
Example 6
Example 5 is repeated, but instead of 7 hours of digestion time, 16 hours of digestion time are applied. 115.1 g of dry astaxanthin are obtained containing 38 mg/kg of dichloromethane.
Example 7
In a variant of example 5, four heating and cooling cycles have been performed during the digestion time of a total of 6 hours (heating from room temperature to 120°C in 20 minutes, keeping 45 minutes at 120 tol25°C, cooling to 35°C in 20 minutes, keeping 15 minutes at 35-28°C then repeat three times this cycle). 110.6 g of dry astaxanthin are obtained containing 267 mg/kg of dichloromethane.
Example 8
An autoclave is inertized with nitrogen, 120 g of AXN and 280 ml of ethyl acetate are introduced into the autoclave, the autoclave is re -inertized with nitrogen and closed, stirring is started (350 rpm), the internal temperature is set up to 119-120°C (reached in ~ 30 min) and kept constant during 4 hours (digestion time) (internal pressure = 3.5 barg). When the desired temperature is reached, beginning of removal of the gas phase and dosing of fresh ethyl acetate to keep the reaction volume constant (flow rate: 2.2 ml per minute).
When the desired digestion time has been applied, the internal temperature is reduced to 20-25°C in ~ 30 min, the autoclave is opened, the slurry is filtered by a Buchner filter. The crystals are washed with 2 x 190 ml of methanol and dried in a drying oven 16 hours at 80°C under 20 mbara. 113.7 g of dry astaxanthin are obtained containing 207 mg/kg of dichloromethane . Example 9
Example 8 is repeated, but instead of 4 hours of digestion time, 7.5 hours of digestion time are applied. Furthermore, the distillate is removed and fresh ethyl acetate are added with a flow rate of 2.7 ml per minute. 118.9 g of dry astaxanthin are obtained containing 80 mg/kg of dichloromethane.
Example 10
In a variant of example 9, removal of the gas phase and dosing of fresh solvent (with a flow rate of 3.0 ml per minute) have been performed only during 10 minutes per hour and during the other 50 minutes per hour the autoclave was closed in each hour of the 6 hours of digestion time. Dry astaxanthin containing 176 mg/kg of dichloromethane is obtained.
Examples 11-12: powdery forms according to the present invention
Example 11 : Astaxanthin 5% Beadlet Form
10 g of purified crystalline Astaxanthin as obtained in example 5 and 1.7 g of dl-a- tocopherol are dissolved in an appropriate solvent. This solution is added under stirring to a solution of 97.7 g of modified food starch (e.g. Capsul HS), 25.0 g of glucose syrup (e.g. Glucidex IT 47), 3.3 g of sodium ascorbate and 240 g of water at 50 to 60°C.This pre-emulsion is homogenized with a rotor-stator-homogenizer for 20 minutes. Eventually the emulsion is homogenized with a high pressure homogenizer. In the next step the remaining solvent is removed by distillation and the solvent- free emulsion is dried by a standard powder catch process. 140 g of beadlets are obtained with an astaxanthin content of 5.7%. Example 12: Astaxanthin 10% Beadlet Form
20 g of purified crystalline Astaxanthin as obtained in example 5 and 1.7 g dl-a- tocopherol are dissolved in an appropriate solvent. This solution is added under stirring to a solution of 81.7 g of modified food starch (e.g. Capsul HS), 13.3 g of glucose syrup (e.g. Glucidex IT 47), 8.3 g of sodium ascorbate and 230 g of water at 50 to 60°C.This pre-emulsion is homogenized with a rotor-stator-homogenizer for 20 minutes. Eventually the emulsion is homogenized with a high pressure homogenizer. In the next step the remaining solvent is removed by distillation and the solvent- free emulsion is dried by a standard powder catch process. 140 g of beadlets are obtained with an astaxanthin content of 12.0%.
Example 13: Astaxanthin 10% Fluid Suspension (an oily suspension according to the present invention)
A coarse suspension which contains 60 g of purified crystalline astaxanthin as obtained in example 5, 5.5 g of dl-a-tocopherol and 480 g of safflower oil is prepared under mixing at 45°C. For particle size reduction, this coarse suspension is pumped continuously through an agitated ball mill (diameter of the zirconiumdioxide pearls: 0.4 mm,
Dispermate SL C12 ball mill; VMA-Getzmann GmbH, Germany) until the desired average particle size (< 2 microns) is achieved. After 140 minutes of milling time, 450 g of an oily suspension results with an astaxanthin content of 10.7% and an average particle size of 1.8 microns (measured with Malvern Mastersizer 3000 equipment, Fraunhofer presentation). Examples 14 and 15: two-fold digestions in ethyl acetate
Example 14
The first digestion was carried out in analogy to the above procedure in ethyl acetate (example 1) with some minor changes: 25.0 g (97.9 wt%) of astaxanthin containing 2214 mg/kg of dichloromethane and 197 mg/kg of methanol, 130 ml of ethyl acetate. Tinternai = 117°C, p = 2.5 barg, time = 16 hours. The oil bath was removed and the suspension was allowed to cool to 40°C for 1 hour (without active cooling) and to 25°C for 1 hour with waterbath-cooling. The filter cake was dried: 16 hours at 60°C (10 mbar). After drying 22.86 g of dark violet astaxanthin crystals were obtained. The crystalline material was subjected to GC head space and HPLC analysis. Results: 150 ppm of methylene chloride (= dichloromethane) and 2 ppm of methanol.
The filter cake from the first digestion was subjected once more to the same procedure: For the second digestion 21.86 g of dried astaxanthin (from the first digestion above) were suspended in 130 ml of ethyl acetate. T (oil bath): 140°C, Tinternai = 117°C, p = 2.6 barg, duration of digestion = 17 hours. After drying for 4 hours at 60°C (10 mbar) and overnight at 120°C (3 x 10~2 mbar) 19.59 g of dark violet astaxanthin crystals were obtained. Analytical results: 44 ppm of methylene chloride (= dichloromethane) and 2 ppm of methanol.
Example 15
The first digestion was carried out in analogy to the above procedure in ethyl acetate (example 1) with some minor changes: 25.0 g (97.9 wt%) of astaxanthin containing 2214 mg/kg of dichloromethane and 197 mg/kg of methanol, 130 ml of ethyl acetate. Tinternai = 119°C, p = 2.9 barg, duration of digestion = 16 hours. The oil bath was removed and the suspension was allowed to cool to 40°C for 1 hour (without active cooling) and to 25°C for 1 hour with waterbath-cooling. Part of the filter cake (1.76 g) was dried: 2.5 hours at 60°C (10 mbar) and 16 hours at 120°C (3 x 10~2 mbar). After drying 1.35 g of dark violet astaxanthin crystals were obtained. The crystalline material was subjected to GC head space and HPLC analysis. Results: 86 ppm of methylene chloride (= dichloromethane) and 2 ppm of methanol.
The rest of the filter cake from the first digestion was subjected once more to the same procedure:
For the second digestion 27.59 g of astaxanthin (from the first digestion above without drying) were suspended in 130 ml of ethyl acetate. T (oil bath): 140°C, Tinternai = 117°C, p = 2.6 barg, duration of digestion = 16 hours. After drying for 5 hours at 60°C (10 mbar) and overnight at 120°C (3 x 10"2 mbar) 18.96 g of dark violet astaxanthin crystals were obtained. Analytical results: 32 ppm of methylene chloride (= dichloromethane) and 2 ppm of methanol.

Claims

Claims
1. A process for the manufacture of synthetic food-grade astaxanthin comprising the following steps:
a) Providing synthetic astaxanthin crystals containing dichloromethane and
optionally methanol; and
b) Adding ethyl acetate to the synthetic astaxanthin crystals in an amount to obtain a suspension of synthetic astaxanthin in ethyl acetate, whereby the amount of synthetic astaxanthin in said suspension is in the range of from 5 to 70 weight-%, based on the total weight of the suspension; and
c) Heating up the suspension obtained in step b) to a temperature in the range of from 110 to 150°C in a closed reactor; and
d) Maintaining the suspension at a temperature in the range as given for step c); and e) Optionally removing residual dichloromethane and ethyl acetate and, if present, methanol by distillation; and
f) Repeating steps b), d) and e) until the content of dichloromethane in the synthetic astaxanthin is < 250 ppm, with the proviso that step b) is only repeated if step e) is performed or repeated; and
g) Cooling the synthetic astaxanthin suspension, whereby its content of
dichloromethane is < 250 ppm, to a temperature in the range of from 10 to 35°C; and
h) Filtering off the synthetic astaxanthin crystals and optionally drying them;
whereby a synthetic food-grade astaxanthin with a content of dichloromethane <
250 ppm is obtained.
A process for the manufacture of synthetic food-grade astaxanthin comprising the following steps:
1) Providing a solution of synthetic astaxanthin in dichloromethane; this solution optionally comprising methanol; and
2) optionally adding methanol to the synthetic astaxanthin solution of step al); a3) removing methanol, if present, and dichloromethane by distillation;
bl) Adding ethyl acetate to the synthetic astaxanthin solution in an amount to obtain a suspension of synthetic astaxanthin, whereby the amount of synthetic astaxanthin in said suspension is in the range of from 5 to 70 weight-%, based on the total weight of the suspension; and
cl) Heating up the suspension obtained in step bl) to a temperature in the range of from 110 to 150°C in a closed reactor; and
dl) Maintaining the suspension at a temperature in the range as given for step cl); and el) Removing residual dichloromethane, ethyl acetate and, if present, methanol by distillation; and
fl) Repeating steps bl), dl) and el) until the content of dichloromethane in the
synthetic astaxanthin is < 250 ppm, with the proviso that step bl) is only repeated if step el) is performed or repeated; and
gl) Cooling the synthetic astaxanthin suspension, whereby its content of
dichloromethane is < 250 ppm, to a temperature in the range of from 10 to 35°C; and
hi) Filtering off the synthetic astaxanthin crystals and optionally drying them;
whereby a synthetic food-grade astaxanthin with a content of dichloromethane < 250 ppm is obtained.
3. The process according to claim 1 or 2, wherein steps b) and e) or steps bl) and el), respectively, are carried out simultaneously.
4. The process according to claim 1 or 2, whereby in step b) or in step bl) the
amount of synthetic astaxanthin in said suspension is in the range of from 20 to 60 weight-%, preferably the amount of synthetic astaxanthin in said suspension is in the range of from 30 to 50 weight-%, based on the total weight of the suspension.
5. The process according to claim 1 or 2 or 4, whereby in step c) or in step cl) the suspension is brought to a temperature in the range of from 110 to 125°C; and in step d) or in step dl) the suspension is then maintained at said temperature.
6. The process according to claim 1 or 2 or 4 or 5, whereby in step g) or in step gl) the synthetic astaxanthin suspension is cooled to a temperature in the range of from 15 to 30°C, preferably to a temperature in the range of from 20 to 25°C.
7. The process according to claim 1 or 2, where the synthetic astaxanthin obtained in step h) or in step hi), respectively, is used as starting material for step a) and step al), respectively.
8. Synthetic food-grade astaxanthin.
9. The synthetic food-grade astaxanthin according to claim 8 being free from
astaxanthin mono- and diesters.
10. The synthetic food-grade astaxanthin according to claim 8 or 9 with a content of dichloromethane < 250 ppm, preferably with a content of dichloromethane < 100 ppm, more preferably with a content of dichloromethane < 10 ppm.
11. The synthetic food-grade astaxanthin according to claim 8 or 9 having a content of methanol < 500 ppm, preferably < 250 ppm, more preferably < 50 ppm.
12. The synthetic food-grade astaxanthin according to claim 10 with a content of
methanol < 500 ppm, preferably with a content of methanol < 250 ppm, more preferably with a content of methanol < 50 ppm.
13. Synthetic food-grade astaxanthin as obtained by any of the processes according to claims 1 to 7.
14. An oily suspension comprising the synthetic food-grade astaxanthin according to any one or more of claims 8 to 13 in an edible oil, said edible oil preferably selected from the group consisting of safflower oil, corn oil, sunflower oil, middle chain triglycerides (MCT) oil and any mixture thereof.
15. The oily suspension according to claim 14, further comprising an antioxidant, whereby preferably the antioxidant is dl-alpha-tocopherol.
16. The oily suspension according to claim 14 and/or 15, wherein the amount of synthetic food-grade astaxanthin in said suspension is in the range of 0.5 to 20 weight-%, based on the total weight of the suspension.
17. A beadlet comprising the synthetic food-grade astaxanthin according to any one or more of claims 8 to 13 encapsulated in a matrix comprising a modified food starch.
18. The beadlet according to claim 17, wherein the amount of synthetic food-grade astaxanthin in said beadlet is in the range of 0.5 to 20 weight-%, based on the total weight of the beadlet.
19. A human dietary supplement comprising synthetic food-grade AXN, free from AXN mono- and diesters, and comprising < 250 ppm of dichloromethane.
20. The human dietary supplement according to claim 19 comprising < 500 ppm of methanol.
21. A method of marketing synthetic food-grade astaxanthin comprising providing a kit said kit comprising
i) synthetic food-grade astaxanthin according to any one or more of claims 8 to 13; ii) information describing the benefits of said synthetic food-grade astaxanthin; iii) optional further information.
22. A method of marketing a human dietary supplement comprising synthetic food- grade AXN, free from AXN mono- and diesters, and comprising < 250 ppm of dichloromethane, comprising providing a kit said kit comprising i) a human dietary supplement comprising synthetic food-grade AXN, free from AXN mono- and diesters, and comprising < 250 ppm of dichloromethane; ii) information describing the benefits of said human dietary supplement;
iii) optional further information.
PCT/EP2014/073956 2013-11-07 2014-11-06 Process for the purification of astaxanthin WO2015067707A1 (en)

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