JP5116982B2 - Method for producing carotenoid - Google Patents

Description

  The present invention relates to a method for producing carotenoids, and more particularly to an industrially suitable method for producing astaxanthin. Specifically, the microorganism culture is extracted with at least one of lower dialkyl ketones and ethers, and then the precipitate obtained by concentrating the extract is treated with at least one of lower alcohols and lower dialkyl ketones or their The present invention relates to a production method for obtaining a composition having a carotenoid content of 90% or more, which contains astaxanthin with a small amount of contaminants derived from a living body, characterized by washing with a mixed solution. The present invention also relates to a carotenoid-containing composition obtained by the method, and further to a food, a pharmaceutical composition, or a cosmetic containing the carotenoid-containing composition.

Carotenoids are natural pigments that exist widely in nature, and are yellow, red, or purple polyene pigments. Astaxanthin is one of the carotenoids found in nature, and exists in a free state or as an ester, and also exists as various chromoproteins in combination with proteins.
Astaxanthin is widely used as a color frying agent for fish and eggs. It is also recognized as a food additive and is widely used in processed oily foods, proteinaceous foods, aqueous liquid foods and the like. Furthermore, from the antioxidant activity against lipid peroxidation induced by free radicals, singlet oxygen scavenging action that reaches several hundred times that of α-tocopherol, etc., functional foods, cosmetics that make use of their strong antioxidant activity, or Expected to be used as a medicine.
Astaxanthin is widely distributed in nature such as salmon, trout, red sea bream fish, crab, shrimp, krill and other crustaceans. It is also produced by microorganisms such as Phaffia yeasts. Carotenoids such as astaxanthin and zeaxanthin are industrially produced by chemical synthesis methods, but those derived from natural products are required because of safety concerns.

In particular, many methods for producing carotenoids containing astaxanthin derived from algae and microorganisms, which are considered to be suitable for mass production, have been reported.
For example, in the case of Haematococcus algae, the cultured algae cyst cells are treated with hot acetone to elute contaminant chlorophyll, and then the cyst cells are spray-dried. From the obtained dried cells, carotenoids are added with ethanol. An extraction method (Patent Document 1) has been reported. However, the composition obtained by such a production method still contains a lot of contaminants derived from living organisms, and is satisfactory in terms of 1) carotenoid content, 2) astaxanthin content, etc. is not.

  In order to obtain a high content of astaxanthin, the crude xanthophyll obtained according to the above method is allowed to act on lipase in the presence of water to decompose neutral lipids, which is one of the contaminants, and the lipase enzyme treatment solution is used. There has been reported a method (Patent Document 2) or the like in which free fatty acid is separated from astaxanthin by distillation from an oil layer separated by oil and water and then purified by distillation. However, even if such a complicated treatment process is performed, an astaxanthin content exceeding 30% has not been obtained.

  In addition, a method for obtaining astaxanthin having a content of 0.5 to 60% by using a supercritical fluid extraction method (Patent Document 3) has also been reported. In order to discard or increase the content, another further concentration operation is required. Therefore, this production method is also unsatisfactory as an industrial method for producing a high-purity carotenoid containing a high amount of astaxanthin containing a small amount of contaminants derived from living organisms in terms of simplicity and economy.

  In addition, as a method using Phaffia genus yeast, a crude oily extract obtained by extracting crushed cells of the yeast with an organic solvent and concentrating the extract is purified by ion exchange chromatography, adsorption chromatography or the like. Although a method for obtaining astaxanthin by carrying out the method (Patent Document 4) has been reported, it is performed by a non-industrial method in which a crude liquid of low concentration astaxanthin is purified by a plurality of column chromatography.

  As another method, a method of producing a crystallization by adding a hydrocarbon solvent to a crude oily substance obtained by extracting cells after culturing the yeast with acetone and concentrating the obtained extract (Patent Document) 5) has been reported. Although this production method is highly convenient, the obtained composition has a carotenoid content of only about 70 to 73% (astaxanthin content is 36 to 42%), and as a method for producing a high-purity carotenoid with little biological impurities. It is not satisfactory.

In addition, in the E-396 strain (FERM BP-4283), from the viewpoint of safety, a method (Patent Document 6) in which a cyclic hydrophilic organic compound that is feared to be used in the production of foods is brought into contact with and extracted from cells. Similar to Patent Document 3, a method using supercritical fluid extraction (Patent Document 7) has been reported. Furthermore, a method of performing liquid-liquid extraction by bringing the E-396 strain into contact with a water-soluble organic solvent, a nonpolar solvent and water has been reported (Patent Document 8).
Under these circumstances, establishment of a method for industrially producing a high-purity carotenoid containing a high content of astaxanthin, which is a simple method that does not require special equipment or complicated operations, has been desired.
JP-A-11-56346 JP 2002-218994 A JP 2004-411147 A JP-A-10-276721 JP 2004-208504 A JP-A-7-242621 JP-A-8-89280 JP-A-8-253695

  An object of the present invention is to provide a high-purity and inexpensive carotenoid-rich composition and an industrial production method thereof, and further to provide a functional food, a pharmaceutical composition, or a cosmetic containing the composition. To do.

As a result of researches focused on microbial cultures in order to solve the above-mentioned problems, the present inventors have obtained 1) more solutions for use in the handling process in order to obtain high-purity carotenoids by the conventional liquid-liquid extraction method. In that respect, it is industrially inefficient, as is the case with the conventional technology for purifying and purifying in a low-concentration solution state, and 2) Solvent recovery with the fractionation operation from the mixed organic solvent is required. Also found a new problem of low convenience. As a result of further earnest research including the solution of the problem, after extracting the microorganism culture with at least one of lower dialkyl ketones and ethers, the extract obtained by concentrating the extract was converted into lower alcohols and lower alcohols. It has been found that a high-purity carotenoid composition can be obtained by washing with at least one dialkyl ketone or a mixture thereof, and the present invention has been completed.
That is, the present invention has the following configuration.
(1) A method for producing a carotenoid-containing composition comprising the following steps 1) to 3):
1) Step of extracting microbial culture with at least one of lower dialkyl ketones and ethers 2) Step of concentrating the resulting extract to obtain a precipitate
3) A method for producing a carotenoid-containing composition comprising the following steps (1) to (3) in which the precipitate is washed with at least one of lower alcohols and lower dialkyl ketones or a mixture thereof.
1) A step of extracting a microorganism culture with acetone 2) A step of concentrating the resulting extract to obtain a precipitate
3) Step for washing precipitate with ethanol (3) A method for producing a carotenoid-containing composition comprising the following steps 1) to 3):
1) Step of extracting the microorganism culture with acetone 2) Step of concentrating the extract obtained to obtain a precipitate 3) Step of washing the precipitate with acetone (4) Carotenoid-containing composition contains 90% or more of carotenoid The manufacturing method in any one of said (1)-(3) which is a composition to contain.
(5) The production method according to any one of (1) to (3), wherein the carotenoid is a carotenoid containing 40% or more of astaxanthin.
(6) The production method according to any one of (1) to (4), wherein the base sequence of DNA corresponding to the 16S ribosomal RNA of the microorganism is substantially homologous to the base sequence set forth in SEQ ID NO: 1 in the Sequence Listing. .
(7) The method for producing carotenoid according to any one of (1) to (5), wherein the microorganism is E-396 strain (FERM BP-4283) or a mutant strain thereof.
(8) A composition containing a carotenoid obtained by the production method according to any one of (1) to (7).
(9) A food, pharmaceutical composition, or cosmetic containing the carotenoid-containing composition according to (8).

  According to the present invention, a highly pure and inexpensive carotenoid-rich composition derived from a natural product and an industrial production method thereof are provided, and further a functional food, a pharmaceutical composition, or a cosmetic containing the composition is provided. be able to.

Hereinafter, the present invention will be specifically described.
The microorganism that can be used in the present invention is not limited as long as it is a microorganism that produces carotenoids, but Paracoccus bacteria, Haematococcus algae, Phaffia yeasts, and the like can be used. In particular, from the viewpoint of the rapid growth rate and the productivity of carotenoids, a bacterium in which the base sequence of DNA corresponding to 16S ribosomal RNA is substantially homologous to the base sequence described in SEQ ID NO: 1 is preferred.
The term “substantially homologous” means that the sequences have 98% or more homology in consideration of the error frequency of DNA base sequence determination. Among these bacteria, E-396 strain (FERM BP-4283) is particularly preferable. In addition, it is also a very preferable example to use a carotenoid high-producing strain selected from the viewpoint of carotenoid productivity by mutation treatment of these microorganisms.

  The microorganism culture used in the present invention is a culture obtained by using a method capable of efficiently culturing the above-mentioned microorganism, for example, a method of culturing by liquid culture, solid culture, or a combination thereof using the following medium. If there is no limit.

As the nutrient medium used for culturing the microorganism used in the present invention, a nutrient medium containing a carbon source, a nitrogen source and an inorganic salt necessary for the growth of the producing bacteria is sufficient, but it is more preferable to add vitamins. There is a case. Further, it may be preferable to further add amino acids, nucleobases and the like. In addition, yeast extract, peptone, meat extract, malt extract, corn steep liquor, dry yeast, soybean meal, etc. may be added as appropriate.
Carbon sources include sugars such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, maltose, acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, pyruvic acid and other organic acids, ethanol, Examples include alcohols such as propanol, butanol, pentanol, hexanol, isobutanol, and glycenol, and fats and oils such as soybean oil, nuka oil, olive oil, corn oil, sesame oil, and linseed oil, and one or more types of carbon A source can be used. The addition ratio varies depending on the type of carbon source and may be adjusted as appropriate, but is usually 1 to 100 g, preferably 2 to 50 g, per liter of the medium.
As the nitrogen source, for example, one or more of potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, urea and the like are used. The addition ratio varies depending on the type of nitrogen source and may be adjusted as appropriate, but is usually 0.1 g to 30 g, preferably 1 to 10 g, with respect to 1 L of the medium.
Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, copper sulfate, chloride One or more of calcium, calcium carbonate, sodium carbonate and the like are used. The addition ratio varies depending on the type of inorganic salt and may be appropriately adjusted, but is usually 0.001 to 10 g with respect to 1 L of the medium.
When adding vitamins, the addition ratio varies depending on the type of vitamins and may be adjusted as appropriate, but is usually 0.1 to 1000 mg, preferably 1 to 100 mg, per 1 L of the medium.
The addition ratio of amino acid, nucleobase, yeast extract, peptone, meat extract, malt extract, corn steep liquor, dry yeast, soybean meal, etc. is different depending on the type of substance and may be adjusted as appropriate. .2 g to 200 g, preferably 3 to 100 g.
The pH of the medium is adjusted to 2-12, preferably 6-9. The culture conditions are 15 to 80 ° C., preferably 20 to 35 ° C., and the culture is usually performed under aerobic conditions for 1 to 20 days, preferably 2 to 8 days. Examples of aerobic conditions include shaking culture or aeration and agitation culture.

  When extracting the astaxanthin produced by the cultured microorganism used in the present invention, after culturing, the culture solution is generally known as a cell concentrate obtained by membrane filtration concentration, or centrifugation, pressure or vacuum filtration, etc. More preferred is a method of subjecting dry bacterial cells obtained by a generally known drying method such as spray drying, fluidized drying, rotary drum drying, or freeze drying to the following extraction: Take an example. In addition, before performing the following extraction, chemical treatment using alkaline reagents or surfactants, lytic enzymes, lipolytic enzymes and It is also possible to perform one or more treatments among biochemical treatments using proteolytic enzymes or the like or physical treatments such as ultrasonic waves or grinding. Normally, when the dried cells are used, about 1 mg of astaxanthin is considered to be contained in 1 g.

Examples of the solvent used for extraction from the cultured microorganism used in the present invention (hereinafter sometimes referred to as an extraction solvent) include at least one of lower dialkyl ketones and ethers, preferably acetone, methyl ethyl ketone, or tetrahydrofuran, acetone, Or tetrahydrofuran is more preferable, and acetone is particularly preferable. The temperature of the solvent during extraction is preferably from 0 ° C. to the boiling point of the solvent, more preferably around 5 ° C. lower than the boiling point of the solvent. In the case of acetone, 25 ° C to 55 ° C is preferable, 40 ° C to 55 ° C is more preferable, and 45 ° C to 52 ° C is further preferable. The amount of the extraction solvent may be an amount that can dissolve the amount of astaxanthin contained in the microbial cells. For example, when extracting from dry microbial cells using acetone, it is 0 with respect to 1 g of astaxanthin contained in the microbial cells. .2-18 kg, preferably 0.9-3 kg.
For example, when extraction from 1 g of dry cells containing about 20 mg of astaxanthin obtained from the above-mentioned cultured microorganism used in the present invention is performed with acetone at 50 ° C., about 7 to 180 g, preferably about 15 to 70 g, more preferably Is preferably used in an amount of about 25 g to 50 g of acetone.

When you want to prevent carotenoid oxidation as much as possible during the extraction operation, select an antioxidant used in an inert gas atmosphere such as nitrogen gas and / or pharmaceuticals and foods and add it to the extraction solvent. Also good.
The antioxidant is preferably finally removed from the carotenoid composition, but depending on the type of antioxidant used, removal is not necessary.
Further, the extraction time is not particularly limited, but when extracting at room temperature or higher, a short time treatment is good in order to reduce a decrease in yield due to thermal decomposition. For example, in the case of using acetone at 50 ° C. Within 12 hours, preferably within 6 hours, more preferably within 3 hours, even more preferably within 2 hours, and most preferably within 1 hour.

Examples of the method for concentrating the extract obtained by extracting the microorganism culture from at least one of lower dialkyl ketones and ethers include heating and / or vacuum concentration. Moreover, in order to prevent the oxidation of carotenoid as much as possible during the concentration, it may be performed in an inert gas atmosphere such as nitrogen gas. The degree of concentration may be appropriately determined in consideration of the amount and purity of the precipitate to be obtained. For example, the concentration may be 3 to 100 times the weight of the extract, preferably 7 to 50 times, Preferably they are 15 times-40 times, Most preferably, they are 20 times-30 times.
Moreover, the distillate obtained by concentrating the extract can be reused for extraction from the microorganism culture as it is.

  The concentrated solution obtained after the concentration is appropriately cooled in consideration of the amount and purity of the resulting precipitate (for example, a temperature that is 20 ° C. or more lower than the temperature during extraction, or 5 ° C., 0 ° C., −5 ° C., −10 ° C. Etc.) to further promote precipitation. Alternatively, after the concentration is stopped immediately before the precipitate is formed, the concentrated solution is appropriately cooled in consideration of the amount and purity of the obtained precipitate (for example, a temperature lower by 20 ° C. or more than the temperature at the time of extraction, or 5 There is also a preferred embodiment in which a precipitate is obtained by heating at 0 ° C., 0 ° C., −5 ° C., −10 ° C. and the like. At this time, seed precipitation may be added for the purpose of smoothly generating the precipitate.

  The precipitate is collected using a vacuum or pressure filter or a centrifuge. At this time, if necessary, the collected precipitate may be washed with a small amount of the same solvent as the extraction solvent. Further, when it is desired to prevent carotenoid oxidation as much as possible, it may be performed in an inert gas atmosphere such as nitrogen gas. Furthermore, considering that the solvent used in the washing of the precipitates to be purified for the subsequent purification will be collected and reused multiple times, the solvent used in the extraction will not remain as much as possible. It is a preferable embodiment to devise a method for collecting precipitates under various conditions.

  In addition, the solvent is recovered from the filtrate containing biological contaminants that are generated when the precipitate is collected, using a method such as distillation or distillation under reduced pressure, and reused for extraction from the microorganism culture and washing of the precipitate. can do.

  In performing the following washing to obtain a high purity carotenoid from the obtained precipitate, an undried product may be used as it is, or a dried product may be used. Further, although the pulverization of the precipitate is not particularly required, a pulverized one may be provided for improving the washing efficiency, or the pulverization may be added during the washing. It is important to use the solvent used for the washing described below, and detailed washing conditions may be appropriately determined according to the purity of the obtained precipitate.

Examples of the solvent used for washing include at least one of lower alcohols and lower dialkyl ketones, or a mixture thereof.
Examples of the lower alcohols include alcohols having 1 to 3 carbon atoms such as methanol, ethanol, n-propanol, and isopropanol. Ethanol or isopropanol is preferable, and ethanol is particularly preferable.
Examples of the lower dialkyl ketones include ketones having 3 to 6 carbon atoms such as acetone, methyl ethyl ketone, diethyl ketone, and methyl isobutyl ketone. Acetone or methyl ethyl ketone is preferable, and acetone is particularly preferable.
Moreover, it is also one preferable aspect to wash with a mixed solvent containing water or another organic solvent to such an extent that the detergency is not reduced.

  As the lower limit amount of the solvent used for washing, it is preferable to select an amount small enough to withstand industrially in consideration of the purity of the precipitate. For example, when ethanol is used, the dried precipitate is washed. When the undried precipitate is washed, a conversion factor that can be set in advance by examining the correlation between the volume or weight of the undried material and the dried precipitate. 2 times or more, but preferably 4 times or more, more preferably 6 times or more, and even more preferably 10 times or more, if necessary. 20 times or more may be used.

  Further, as the upper limit amount, it is sufficient that the amount is 200 times at the maximum, particularly 100 times or less is preferable from the viewpoint of industrial efficiency, 80 times or less is more preferable, and 40 times or less is more preferable. Is set to be 20 times or less, and in some cases, about 10 times is sufficient.

  For example, when using acetone, when washing the dried precipitate, it is based on its weight; when washing the dried precipitate, the volume or weight of the dried substance and the dried precipitate are used. 30 weight or more, preferably 35 times or more, more preferably 35 times or more of the dry matter weight calculated using a set conversion factor that can be set by considering the correlation with The amount is 40 times or more, and if necessary, 40 times or more may be used. Further, as the upper limit amount, it is sufficient that the amount is 200 times at the maximum, and in particular, from the viewpoint of industrial efficiency, it is preferably 100 times or less, more preferably 80 times or less, still more preferably 60 times or less, If it is set in detail, about 50 times or less is sufficient.

  The method of washing is not limited at all, but, for example, a method of filtering after suspension and stirring, a method of passing liquid from above the precipitate, and the like are practically preferable methods. The temperature at the time of washing is usually preferably 1 ° C. to 30 ° C., but may be 1 ° C. or lower or 30 ° C. or higher depending on the situation. However, the upper limit temperature includes the vicinity of the boiling point of the solvent used for washing (for example, 78 ° C. for ethanol and 56 ° C. for acetone). Further, when it is desired to prevent carotenoid oxidation as much as possible, it may be performed in an inert gas atmosphere such as nitrogen gas.

Moreover, the solvent used by methods, such as distillation and depressurizing distillation, can be collect | recovered from the waste liquid generate | occur | produced by the washing | cleaning for obtaining a high purity carotide from a deposit, and it can reuse as a solvent used for washing | cleaning.
In addition, for the purpose of reducing the amount of residual solvent in the carotenoid-containing composition of the present invention after washing and drying, it is very difficult to add a step of washing with water at the end of washing as needed. This is a preferable method. For example, a method of adding a step of washing with a small amount of low-temperature water or ethanol at the end is a preferred example.

Adjustment of the carotenoid content in the carotenoid-containing composition obtained by using the above production method and the content of main components such as astaxanthin may be appropriately changed so as to maximize the yield.
The carotenoid content in the carotenoid-containing composition of the present invention is preferably 90% or more, more preferably 91% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 98% or more.
Further, the astaxanthin content in the carotenoid is preferably 40% or more, more preferably 45% or more, further preferably 46% or more, particularly preferably 47% or more, particularly preferably 50% or more, and particularly preferably 55% or more. Very much preferred, with 60% or more being most preferred.
Specifically, for example, by extracting the microorganism culture with acetone and then washing the precipitate obtained by concentrating the extract with ethanol, a composition having a carotenoid content of 95% or more can be obtained. After the microorganism culture is extracted with acetone, the precipitate obtained by concentrating the extract is washed with acetone, whereby a composition having a carotenoid content of 90% or more can be obtained.

In the production method of the present invention, after extracting at least one of lower dialkyl ketones and ethers from a microorganism culture, the precipitate obtained by concentrating the extract is converted into an alcohol-containing solvent having 1 to 3 carbon atoms or 3 carbon atoms. It is characterized in that the carotenoid can be highly purified only by an extremely simple operation of washing with a -6 ketone-containing solvent.
Compared with the prior art, the method of the present invention 1) does not require complicated operations, and 2) is inefficient and high in a low concentration solution of 1% or less such as column purification or liquid-liquid extraction. This is a significant advantage from an industrial point of view in that no purification operation for purification is required. And as an effect of the present invention, 3) a high-purity carotenoid composition containing astaxanthin in a high content can be provided at low cost, and 4) an excellent industrial production method in that the solvent used in each step can be easily recovered. It is to provide.

A food, pharmaceutical composition, or cosmetic containing the carotenoid-containing composition of the present invention is also within the scope of the present invention.
Examples of pharmaceutical forms containing high-purity carotenoids with high astaxanthin content produced by the production method of the present invention include scattered, granule, pill, soft capsule, hard capsule, tablet, chewable tablet, quick-disintegrating tablet, syrup, liquid preparation , Suspensions, suppositories, ointments, creams, gels, sticking agents, inhalants, injections and the like. These preparations are prepared according to conventional methods. However, since carotenoids are sparingly soluble in water, they can be dissolved in non-hydrophilic organic solvents such as vegetable oils and animal oils, or emulsifiers, dispersed or surfactants. And the like, and dispersed and emulsified in an aqueous solution using a homogenizer (high pressure homogenizer). Furthermore, in order to increase the absorbability of carotenoids, the average particle system can be finely pulverized to about 1 micron.

  Additives that can be used for formulation include animal and vegetable oils such as soybean oil, safflower oil, olive oil, germ oil, sunflower oil, beef tallow, sardine oil, polyethylene glycol, propylene glycol, glycerin, sorbitol, etc. Surfactant such as polyhydric alcohol, sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, purified water, lactose, starch, crystalline cellulose, D-mannitol, lecithin, gum arabic, sorbitol solution, sugar Examples thereof include excipients such as liquid, sweeteners, colorants, pH adjusters, and fragrances. The liquid preparation may be dissolved or suspended in water or other appropriate medium when taken. Tablets and granules may be coated by a known method.

  When administered in the form of an injection, it can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, transdermally, intraarticularly, within the bursa, intravesicular, intraperiosteally, sublingually, orally. In particular, intravenous administration or intraperitoneal administration is preferred. Intravenous administration may be either drip administration or porous administration.

  Examples of the form of the food containing the high-purity carotenoid having a high astaxanthin content of the present invention include supplements (scattering, granules, soft capsules, hard capsules, tablets, chewable tablets, quick-disintegrating tablets, syrups, liquids, etc.), beverages (tea, tea, Carbonated drinks, lactic acid drinks, sports drinks, etc.), confectionery (gummy, jelly, gum, chocolate, cookies, candy, etc.), oil, oil and fat foods (mayonnaise, dressing, butter, cream, margarine, etc.), ketchup, sauce, liquid food Dairy products (milk, yogurt, cheese, etc.), breads, noodles (udon, soba, ramen, pasta, yakisoba, kishimen, somen, hiyagigi, rice noodles, etc.).

  As functional food containing high purity carotenoid with high astaxanthin content of the present invention, various nutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin E, etc.), if necessary, Various minerals, dietary fiber, polyunsaturated fatty acids, other nutrients (coenzyme Q10, carnitine, sesamin, α-lipoic acid, inositol, D-kaileunositol, pinitol, phosphatidylserine, phosphatidylserine DHA, phosphatidylinositol, taurine, glucosamine , Chondroitin sulfate, S-adnosylmethionine, etc.), dispersants, stabilizers such as emulsifiers, sweeteners, taste ingredients (citric acid, malic acid, etc.), flavors, royal jelly, propolis, agarics, etc. it can. Moreover, you may mix | blend herbs, such as peppermint, bergamot, camomile meal, and lavender. Moreover, you may mix | blend raw materials, such as theanine, a dehydroepiandosterone, and melatonin.

  Examples of cosmetics containing a high-purity carotenoid with a high astaxanthin content of the present invention include creams, emulsions, lotions, microemulsion essences, bathing agents, and the like, and fragrances and the like may be mixed.

Although this invention is demonstrated based on an Example, a reference example, a formulation example, and a test example, the scope of the present invention is not limited to the following examples.
In the examples and comparative examples, astaxanthin and carotenoids were quantified using high performance liquid chromatography (HPLC). As the column, two Wakosil-II SIL-100 (φ4.6 × 250 mm) (manufactured by Wako Pure Chemical Industries, Ltd.) were connected and used. The elution was performed by flowing 1.0 mL / min of a mobile phase n-hexane-tetrahydrofuran-methanol mixture (40: 20: 1) at a constant temperature near room temperature. In the measurement, 20 μL of a solution obtained by dissolving a sample in tetrahydrofuran with 100-fold dilution in a mobile phase was used as the injection amount, and the column eluent was detected at a wavelength of 470 nm. In addition, astaxanthin (Cat. No. A9335) manufactured by Sigma was used as a standard product for quantification. The astaxanthin concentration of the standard solution was set using the following equation after measuring the absorbance at 477 nm of the standard solution (A) and the area percentage% (B) of the astaxanthin peak when HPLC analysis was performed under the above conditions. It was.
Astaxanthin concentration (mg / L) = A ÷ 2150 × B × 100

[Example 1] Production 1 of high-purity carotenoid with high astaxanthin content
Step 1: Culture process of E-396 strain 10 ml of a medium consisting of glucose 2 g / L, meat extract 3 g / L, peptone 10 g / L, and sodium chloride 5 g / L is placed in a test tube having a diameter of 18 mm, Steam sterilized for minutes. E-396 strain (FERM BP-4283) was inoculated with 1 platinum ear, and reciprocal shaking culture at 300 rpm was performed at 30 ° C. for 6 days. 200 culture solutions (2 L) were centrifuged and then lyophilized to obtain dry cells containing 18 mg of astaxanthin in 1 g.

Step 2: Acetone extraction step 2.2 kg of acetone was added to 50 g of the dried cells obtained in Step 1 of Example 1, and carotenoids including astaxanthin were extracted while stirring at 50 ° C for 1 hour. Next, the microbial cells are removed by filtration, and the microbial cake is further washed with acetone to extract 2.3 kg of an astaxanthin 0.034% (wt / wt) and carotenoid weight concentration 0.077% (wt / wt). Got.

Step 3: Extraction liquid concentration and precipitation process 2.3 kg of the extraction liquid obtained in Step 2 of Example 1 was concentrated under reduced pressure using an evaporator, and a concentrated liquid (about 80 g) containing a precipitate and a distillate. (About 2 kg of acetone) was obtained. The concentrate was stirred while cooling at an external temperature of 5 ° C. for 1 hour.

Step 4: Precipitate filtration and drying step About 80 g of the cooled concentrated liquid obtained in Step 3 of Example 1 was filtered under reduced pressure to obtain a precipitate cake containing carotenoids. Further, 20 g of acetone was added from above and washed by filtration, followed by drying under reduced pressure at 35 ° C. overnight to obtain 2.13 g of a dried precipitate. The contents of astaxanthin and carotenoid in the dried product were 34% and 71%, respectively.

Step 5: Ethanol washing step and drying step 80 g of ethanol was added to 2.13 g of the dried precipitate obtained in Step 4 of Example 1, and 1.8% of the carotenoid was suspended at room temperature. Washing with stirring was performed for 1 hour. The obtained washed product was collected by vacuum filtration and dried overnight at 35 ° C. to obtain 1.49 g of ethanol washed product. The contents of astaxanthin and carotenoid in this ethanol-washed and dried product were 47% and 98%, respectively.

Step 6: Recovery of solvent by distillation under reduced pressure From about 100 g (about 120 mL) of the filtrate generated in Step 4, 80 g of acetone was recovered on the side of the distillation distillation by distillation under reduced pressure. In addition, 76 g of ethanol was recovered on the distillate side from about 80 g (about 100 mL) of the filtrate generated in Step 5 by distillation under reduced pressure.

[Example 2] Production 1 of high-purity carotenoid with high astaxanthin content using recovered solvent 1
1.1 kg of the distilled liquid (acetone) obtained in Step 3 of Example 1 was added to 25 g of the dried cells obtained in Step 1 of Example 1, and carotenoids were extracted in accordance with Step 2 of Example 1. Bacterial cell filtration was performed to obtain 1.2 kg of an extract. The extract was concentrated under reduced pressure according to Step 3 of Example 1, and then about 40 g of a cooled concentrate was obtained. A precipitate was collected from the cooled concentrate according to Step 4 of Example 1. Next, 10 g of acetone collected in Step 6 of Example 1 was added from above the precipitate cake, washed by filtration, and then dried to obtain 1.00 g of a dried product. To this dried product, 40 g of ethanol recovered in Step 6 of Example 1 was added, and the same procedure as in Step 5 of Example 1 was performed to obtain 0.72 g of ethanol-dried product. The contents of astaxanthin and carotenoid in the dried product under reduced pressure before ethanol washing were 33% and 70%, respectively, whereas the contents of astaxanthin and carotenoid in the ethanol washed product were 46% and 97%, respectively.
From the above, it was confirmed that there was no problem in reusing the recovered solvent.

[Example 3] Production of high purity carotenoid with high astaxanthin content 2
In the same manner as in Step 1 to Step 3 of Example 1, and further according to Step 4 of Example 1, about 80 g of the cooled concentrated liquid obtained in Step 3 was filtered under reduced pressure to obtain a carotenoid-containing precipitate. Got a cake. Further, 20 g of acetone was added from the top, and after filtering and washing, a portion of the cake was collected to examine the carotenoid content of the precipitate. Next, the drying step was omitted, and 80 g of ethanol was added from the top and further washed by filtration. This filter washing cake was dried under reduced pressure at 35 ° C. overnight to obtain 1.50 g of a dried ethanol washed product. The contents of astaxanthin and carotenoid in the vacuum dried product before washing with ethanol were 33% and 70%, respectively, whereas the contents of astaxanthin and carotenoid in the ethanol washed product were 46% and 97%, respectively. .
Further, from about 100 g (about 120 mL) of the filtrate obtained by the precipitate filtration performed in accordance with Step 4 of Example 1, 85 g of acetone is obtained in the ethanol washing step performed in accordance with Step 5 of Example 1. From about 80 g (about 100 mL) of the filtrate, 75 g of ethanol slightly mixed with acetone was recovered.

[Example 4] Production of high-purity carotenoid with high content of astaxanthin using recovered solvent 2
To 25 g of dry cells used in Example 3, 1.1 kg of the distillate obtained in Example 3 (acetone) was added, followed by carotenoid extraction and cell filtration according to Step 2 of Example 1, 1.1 kg of extract was obtained. The extract was concentrated under reduced pressure according to Step 3 of Example 1, and then about 40 g of a cooled concentrate was obtained. A precipitate was collected from the cooled concentrate according to Step 4 of Example 1. Next, 10 g of the recovered acetone obtained in Example 3 was added from above the cake of the precipitate, washed by filtration, and further dried in the same manner as in Example 3, omitting 40 g of ethanol recovered in Example 3. Was further filtered and washed. This filtration washing cake was dried under reduced pressure at 35 ° C. overnight to obtain 0.73 g of a dried ethanol washed product. The contents of astaxanthin and carotenoid in the vacuum-dried product before washing with ethanol were 34% and 71%, respectively, whereas the contents of astaxanthin and carotenoid in the ethanol-washing dried product were 46% and 97%, respectively.
It was confirmed that even if ethanol mixed with acetone collected by distillation under reduced pressure in Example 4 was used, no reduction in cleaning ability was observed. However, in consideration of collecting and reusing a plurality of times, it is considered preferable to filter the precipitate under conditions such that acetone does not remain as much as possible before the ethanol washing step.

[Example 5] Production of high-purity carotenoid with high astaxanthin content 3
The same procedure was performed except that acetone cleaning was performed instead of ethanol cleaning in Example 1. As a result, the contents of astaxanthin and carotenoid in the obtained dried product were 45% and 91%, respectively.

[Reference Example 1] Measurement of dissolved concentration of astaxanthin at room temperature The dissolved concentration of astaxanthin (manufactured by Sigma) at room temperature was measured and shown in Table 1.
As is clear from Table 1, it was found that the dissolution concentration in solvents other than halogen solvents having carcinogenicity and mutagenicity was as low as 1% or less. Therefore, as long as a general organic solvent that is relatively less harmful in the liquid-liquid distribution of the prior art is used, it is extremely difficult to carry out a high-purity treatment with a solution having a carotenoid concentration exceeding 1%. It became clear that there was a great problem in industrial productivity.

[Comparative Example 1] Production method according to Example 1-1 of JP-A-8-253695 A astaxanthin concentration of 0.034% (wt / wt) and carotenoid concentration obtained in the same manner as in Steps 1 and 2 of Example 1 To 2.3 kg (2.9 L) of 0.077% (wt / wt) extract, 1.9 kg (2.9 L) of hexane and 2.9 kg (2.9 L) of 1% saline were added and stirred for 1 hour. . After standing, two layers were separated to obtain 3.7 L (2.5 kg) of the upper hexane layer and 5 L (4.6 kg) of the lower aqueous layer. The obtained hexane layer was concentrated under reduced pressure using an evaporator to prepare a concentrated solution of about 0.3 L, and left at 4 ° C. for 12 hours, and then the precipitate was filtered under reduced pressure, and 25 mL of hexane was added from above and washed by filtration. And dried overnight under reduced pressure to obtain 1.45 g of a dried product having an astaxanthin content of 47% and a carotenoid content of 98%.

For example, in Example 1, about 1.8% (wt / wt) carotenoid suspension can be handled under the conditions for the high purity treatment of carotenoid, while in Comparative Example 1, the carotenoid concentration is about 0.07. It was necessary to contact a 5 L aqueous layer with 3.7 L of a hexane solution having a low carotenoid concentration of% (wt / wt). From this point, it was revealed that the method of Comparative Example 1 is industrially inefficient.
Also, in terms of the total amount of liquid handled, for example, the total volume of acetone and hexane and 1% saline used in Comparative Example 1 is 8 compared to 2.925 L of the total volume of acetone and ethanol used in Example 1. Since it was .725L, the handling amount of the process liquid was about three times as large. From this point, it became clear that it is industrially inefficient.
Furthermore, about 2.9 L of acetone used for extraction was recovered, for example, in Examples 1 and 3, although about 2.8 L of acetone was recovered as a distillate simultaneously with the vacuum concentration of the extract during the production process. On the other hand, in Comparative Example 1, the operation of recovering acetone and hexane respectively by newly fractionating from about 3.4 L of a distillate consisting of acetone and hexane obtained by vacuum concentration of the hexane layer and about 5 L of water layer An operation for newly collecting acetone was required. This also revealed that the convenience of the solvent recovery process is low.

[Food Example 1] Margarine As an antioxidant and colorant, the astaxanthin composition obtained in Example 1 was added to vegetable oil so as to be 5% by weight of margarine, and then stirred uniformly with an emulsifier. Margarine was prepared by a conventional method. This margarine had a light red color due to the presence of astaxanthin as compared to normal margarine.

[Food example 2] Artificial salmon roe Addition of 0.6% of the astaxanthin composition obtained in Example 1 to a 1% aqueous sodium alginate solution, disperse with a homogenizer, drop it into a 5% calcium chloride coagulation solution, and a spherical shape with a diameter of 5 mm Molded into. The appearance was close to nature and was very similar to salmon roe in terms of shape, color and taste.

[Formulation Example 1] Astaxanthin-containing tablet With respect to 120 parts by weight of the carotenoid-containing composition obtained in Example 1, 330 parts by weight of crystalline cellulose, 15 parts by weight of carmellose-calcium, 10 parts by weight of hydroxypropyl cellulose and 60 parts by weight of purified water After mixing and drying by a usual method, 10 parts by weight of magnesium stearate was added and tableted to obtain 100 mg tablets containing 20 mg of carotenoid-containing composition per tablet.

[Formulation example 2] Astaxanthin-containing soft capsules Suspended in 1 part by weight of the carotenoid-containing composition obtained in Example 1 in 5 times by weight of soybean oil and mixed well so as to be homogeneous, and then in a capsule filling machine Capsules were filled to obtain a reddish brown capsule having a content of about 300 mg.

[Cosmetics] Astaxanthin-containing cream (cosmetics)
The astaxanthin-containing composition obtained in Example 1 was added to white petrolatum so as to be 10% by weight and stirred uniformly with a fragrance and the like to prepare a cream by a usual method.

  According to the present invention, it is possible to provide a highly pure and inexpensive carotenoid-rich composition derived from a natural product and an industrial production method thereof, and as a result, a functional food and a pharmaceutical composition containing the composition. Or cosmetics can be provided.

Claims (5)

A method for producing a carotenoid-containing composition containing 90% or more of carotenoid, comprising the following steps 1) to 3), wherein the carotenoid contains 40% or more of astaxanthin.
1) Extracting a culture of Paracoccus bacteria with at least one selected from acetone, methyl ethyl ketone and tetrahydrofuran at 25 ° C. to 55 ° C. 2) Concentrating the resulting extract to obtain a precipitate 3) Converting the precipitate to carbon A step of washing with at least one kind of alcohol having 1 to 3 alcohols and ketone having 3 to 6 carbon atoms or a mixture thereof. A method for producing a carotenoid-containing composition containing 90% or more of carotenoid, comprising the following steps 1) to 3), wherein the carotenoid contains 40% or more of astaxanthin.
1) Step of extracting a culture of Paracoccus bacteria with acetone at 25 ° C. to 55 ° C. 2) Step of concentrating the obtained extract to obtain a precipitate 3) Step of washing the precipitate with ethanol A method for producing a carotenoid-containing composition containing 90% or more of carotenoid, comprising the following steps 1) to 3), wherein the carotenoid contains 40% or more of astaxanthin.
1) Step of extracting a culture of Paracoccus bacteria with acetone at 25 ° C. to 55 ° C. 2) Step of concentrating the obtained extract to obtain a precipitate 3) Step of washing the precipitate with acetone   The production method according to any one of claims 1 to 3, wherein the base sequence of DNA corresponding to 16S ribosomal RNA of a Paracoccus bacterium has 98% or more identity with the base sequence set forth in SEQ ID NO: 1 in Sequence Listing.   The method for producing a carotenoid according to any one of claims 1 to 3, wherein the Paracoccus bacterium is E-396 strain (FERM BP-4283).