Methods for isomerizing trans xanthophylls

Abstract

To provide a method for isomerizing novel trans-form xanthophyll into cis-form xanthophyll.SOLUTION: A method for isomerizing trans-form xanthophyll into cis-form xanthophyll includes: a process of bringing microorganism containing the trans-form xanthophyll in contact with fluid containing organic solvent, in which the solubility of astaxanthin in supercritical CO2 and at 25°C is 0.001 g / L to 0.4 g / L, or fluid containing carbon number 1 to 6 of alcohol under the temperature condition of 70 to 160°C.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for isomerizing trans-xanthophyll, and more particularly to a method for isomerizing trans-xanthophyll into cis-xanthophyll.

Background Art

[0002] Xanthophyll is a natural pigment widely present in nature. Known types of xanthophyll include astaxanthin, adonirubin, adonixanthin, zeaxanthin, and β-cryptoxanthin. It is known that astaxanthin, adonirubin, adonixanthin, etc. have various effects including an anxiolytic physiological effect (Patent Document 1). Therefore, astaxanthin, adonirubin, adonixanthin, etc. are expected to be used as foods, pharmaceuticals, etc.

[0003] Astaxanthin, adonirubin, adonixanthin, etc. are widely distributed in animals, plants, and microorganisms. For example, astaxanthin is widely distributed in nature such as fish such as salmon, trout, and sea bream, and crustaceans such as crabs, shrimps, and krill. Also, astaxanthin is produced by bacteria belonging to the genera Agrobacterium, Brevibacterium, Paracoccus, Brevundimonas, and Erythrobacter, green algae belonging to the genus Haematococcus, yeasts belonging to the genus Phaffia, etc. Xanthophylls such as astaxanthin and adonixanthin are industrially produced by chemical synthesis methods, but due to the increasing health consciousness and environmental protection awareness, those derived from natural products are in demand.

[0004] Among the microorganisms mentioned above, bacteria belonging to the genus Paracoccus have advantages such as high xanthophyll productivity and rapid growth rate. An example of an astaxanthin-producing strain belonging to the genus Paracoccus is strain E-396 (FERM BP-4283: April 27, 1993 (original deposit date), National Institute of Advanced Industrial Science and Technology Patent Organism Depository Center (1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan)) (Patent Document 2).

[0005] On the other hand, cis-isomerization of xanthophyll is known to contribute to improved extraction efficiency from bacterial cells, and cis-isomers of xanthophyll are also known to contribute to improved absorption in the body. However, when attempting to obtain cis-type xanthophyll from microorganisms, xanthophyll contained in microbial cells has a problem of low isomerization efficiency and low yield compared to isomerizing the xanthophyll molecule itself, due to factors such as uptake into cellular tissues like the cell membrane and interaction with biomacromolecules such as lipids contained in the cell.

[0006] Here, Patent Document 3 describes a method for producing tomato extract in which the cis-lycopene content is increased by further heating the lycopene-containing extract obtained by supercritical CO2 extraction from tomatoes or tomato products at a high temperature. However, the method described in Patent Document 3 is a method for increasing the cis-lycopene content by heating the extract obtained by supercritical CO2 extraction at a high temperature (110°C to 140°C), and astaxanthin is not mentioned. Astaxanthin is a carotenoid (particularly called xanthophyll) that has a polar hydroxyl group, and is known to have low solubility in nonpolar molecules such as CO2, resulting in low extraction efficiency by supercritical CO2 extraction. Furthermore, paragraph 0019 of Patent Document 3 states that by employing supercritical extraction, organic solvents become unnecessary.

[0007] Patent Document 4 describes a method for separating and purifying astaxanthin from cultured Phaffia rhodozyma cells, characterized by the combined use of supercritical CO2 extraction with ethanol as a cosolvent and silver nitrate aqueous solution extraction. However, the method described in Patent Document 4 involves crushing the cultured cells, pre-treating them with an organic solvent and silver nitrate, and then subjecting them to supercritical CO2 extraction, rather than directly extracting xanthophyll from the cells. Furthermore, there is no mention of supercritical CO2 extraction at high temperatures, cis-trans isomerization, or content ratio.

[0008] Patent Document 5 describes a method for producing an astaxanthin concentrate by degreasing and concentrating a natural product-derived raw material (Haematococcus) containing astaxanthin using supercritical countercurrent contact extraction in a CO2-ethanol mixed system. However, the method described in Patent Document 5 refers to supercritical CO2 extraction of astaxanthin derived from Haematococcus, and the origin of the astaxanthin is different from that of the present invention. Furthermore, in the method described in Patent Document 5, dried Haematococcus is added to ethanol, and the insoluble matter is filtered out and subjected to supercritical CO2 extraction; astaxanthin is not extracted directly from the dried material. Moreover, astaxanthin derived from Haematococcus is specifically in the ester form, while astaxanthin derived from Paracoccus bacteria exists as a free form. Free form astaxanthin is known to have lower solubility and be more difficult to extract than ester form astaxanthin. Furthermore, the method described in Patent Document 5 has a low astaxanthin recovery rate from raw materials of 10% or less, and there is no mention of the cis-trans isomerization rate.

[0009] Therefore, from the perspective of improving extraction efficiency from microorganisms and enhancing absorption in the body, there is still a need for isomerization technology for cis-type xanthophylls that can be isomerized while still contained within microorganisms. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2012-025712 [Patent Document 2] Japanese Patent Application Publication No. 08-009964 [Patent Document 3] Japanese Patent Publication No. 2017-019756 [Patent Document 4] Japanese Patent Application Publication No. 08-140695 [Patent Document 5] Japanese Patent Publication No. 2018-198549 [Overview of the Initiative]

[0011] The present inventors have now discovered that xanthophylls with a high proportion of the cis isomer can be efficiently extracted by contacting microorganisms containing trans xanthophylls with supercritical CO2 at high temperatures and a fluid containing an organic solvent in which astaxanthin has a solubility of 0.001 g / L to 0.4 g / L at 25°C, or a fluid containing an alcohol with 1 to 6 carbon atoms. The present invention is based on this finding.

[0012] Therefore, the present invention provides a method for improving the cis-isomer ratio of microorganisms containing trans-type xanthophyll.

[0013] The present invention encompasses the following inventions. [1] A method for isomerizing trans xanthophyll to cis xanthophyll, comprising the step of contacting a microorganism containing the trans xanthophyll with a fluid containing supercritical CO2 and an organic solvent having a solubility of astaxanthin of 0.001 g / L to 0.4 g / L at 25°C, under temperature conditions of 70 to 160°C. [2] A method for isomerizing trans xanthophyll to cis xanthophyll, comprising the step of contacting a microorganism containing the trans xanthophyll with a fluid containing supercritical CO2 and an alcohol having 1 to 6 carbon atoms under temperature conditions of 70 to 160°C. [3] The method according to [2], wherein the alcohol is ethanol. [4] The method according to any one of [1] to [3], wherein the temperature condition is 80°C to 140°C. [5] The method according to any one of [1] to [4], wherein the pressure conditions in the contact step with the fluid are 20 MPa or higher. [6] The method according to any one of [1] to [5], wherein the concentration of the organic solvent or the alcohol is 15% to 50% by mass with respect to the supercritical CO2. [7] The method according to any one of [1] to [6], wherein the microorganism is Paracoccus carotinifaciens. [8] The method according to any one of [1] to [7], wherein the trans xanthophyll is at least one selected from the group consisting of trans astaxanthin, trans adonirubin, trans adonixanthin, trans zeaxanthin, and trans β-cryptoxanthin. [9] A method for producing a cis-xanthophyll-containing composition, comprising the step of contacting a microorganism containing trans-xanthophyll with a fluid containing supercritical CO2 and an organic solvent having a solubility of astaxanthin of 0.001 g / L to 0.4 g / L at 25°C, under temperature conditions of 70 to 160°C.

[10] A method for producing a cis-type xanthophyll-containing composition, comprising the step of contacting a microorganism containing trans-type xanthophyll with a fluid containing supercritical CO2 and an alcohol having 1 to 6 carbon atoms under temperature conditions of 70 to 160°C.

[11] The method according to [9] or

[10] , wherein the composition comprises at least one selected from the group consisting of cis-astaxanthin, cis-adonilbin, and cis-adonixanthin.

[12] The method according to

[11] , wherein the proportion of the cis isomer of astaxanthin in the composition is 22 area% or more, the proportion of the cis isomer of adonilubin is 24 area% or more, or the proportion of the cis isomer of adonixanthin is 22 area% or more.

[13] The method according to any one of [9] to

[12] , wherein the microorganism is Paracoccus carotinifaciens.

[14] A cis-xanthophyll-containing composition comprising at least one selected from the group consisting of cis-astaxanthin, cis-adonirubin, and cis-adonixanthin, the composition satisfying at least any one of the following (i) to (iii): (i) The cis-isomer ratio of endogenous astaxanthin is 51 area% or more. (ii) The cis-isomer ratio of endogenous adonirubin is 57 area% or more. (iii) The cis-isomer ratio of endogenous adonixanthin is 47 area% or more.

[15] The composition according to

[14] , wherein all the xanthophylls in the composition are endogenous.

[16] The composition according to

[14] or

[15] , wherein the xanthophyll in the composition is in a free form.

[17] The composition according to any one of

[14] to

[16] , wherein the composition is a dried product.

[0014] According to the present invention, trans-xanthophyll can be efficiently isomerized to cis-xanthophyll. Therefore, the cis-isomer ratio of a microorganism containing trans-xanthophyll can be improved. Also, according to the present invention, cis-xanthophyll can be recovered from a microorganism containing trans-xanthophyll with a high yield. Furthermore, according to the present invention, at least any one of the recovery rate, cis-isomer ratio, and cis-isomer yield of xanthophyll in the cis-xanthophyll-containing composition can be improved. Also, according to the present invention, in the cis-xanthophyll-containing composition, it is advantageous in that the endogenous cis-isomer ratio can be improved. '

Brief Description of the Drawings

[0015] [Figure 1]FIG. 1 is a schematic diagram of a supercritical extraction apparatus used in a method for isomerizing trans-xanthophyll to cis-xanthophyll according to the present invention. Detailed description of the invention

[0016] The method for isomerizing trans-xanthophyll to cis-xanthophyll according to the present invention includes a step of contacting a microorganism containing the trans-xanthophyll with a fluid containing supercritical CO2 and an organic solvent having a solubility of astaxanthin at 25°C of 0.001 g / L to 0.4 g / L or a fluid containing an alcohol having 1 to 6 carbon atoms under a temperature condition of 70 to 160°C, which is one of the features.

[0017] In this specification, for a description using "~" for a numerical range, unless otherwise specified, it includes the lower limit value and the upper limit value. For example, in the description of "10~20", it includes both the lower limit value of "10" and the upper limit value of "20". That is, "10~20" has the same meaning as "10 or more and 20 or less".

[0018] Xanthophyll According to an embodiment of the present invention, xanthophyll is not particularly limited as long as it does not interfere with the effects of the present invention. Specific examples of xanthophyll in the present invention include astaxanthin, adonirubin, adonixanthin, zeaxanthin, β-cryptoxanthin, lutein, echinenone, canthaxanthin, 3-hydroxyechinenone, asteroidenone, violaxanthin, anteraxanthin, neoxanthin, fucoxanthin, peridinin, and rhodoxanthin, etc. Preferably, they are astaxanthin, adonirubin, and adonixanthin. These xanthophylls may be used alone or in combination of two or more. Such xanthophyll may be, for example, a combination of astaxanthin, adonirubin, and adonixanthin.

[0019] According to another embodiment of the present invention, it is preferable that the xanthophyll further comprises at least one selected from the group consisting of canthaxanthin, asteroidenone, echinenone, and 3-hydroxyechinenone, in addition to astaxanthin, adonirubin, and adonixanthin. It is even more preferable that the xanthophyll further comprises canthaxanthin, asteroidenone, echinenone, and 3-hydroxyechinenone, in addition to astaxanthin, adonirubin, and adonixanthin.

[0020] Xanthophylls have isomers based on the cis and trans conjugated double bonds in the central part of the molecule. Isomers in which one or more conjugated double bonds in the molecule are in the cis position are called "cis xanthophylls," while isomers in which all conjugated double bonds in the molecule are in the trans position are called "trans xanthophylls," "trans xanthophylls," or "all-trans xanthophylls." When simply referred to as "xanthophylls," both cis and trans xanthophylls are included. The definitions of cis and trans isomers also apply to each xanthophyll, such as astaxanthin, adonirubin, and adonixanthin.

[0021] Astaxanthin is a red pigment, and its chemical formula is 3,3'-dihydroxy-β,β-carotene-4,4'-dione (C 40 H 52 04, molecular weight 596.852). The structural formula of trans astaxanthin is represented by the following formula (A). In addition, cis astaxanthin includes the 9-cis, 13-cis, 15-cis, dicis, or combinations thereof. Here, the structural formula of 9Z-astaxanthin is represented by the following formula (B), and the structural formula of 13Z-astaxanthin is represented by the following formula (C). [ka]

[0022] The chemical formula for adonilvin is 3-hydroxy-β,β-carotene-4,4'-dione (C 40 H 52 (03, molecular weight 580.853), and the structural formula of trans-adonirubin is represented by the following formula. In addition, cis-adonirubin includes cis isomers such as 9-cis, 13-cis, and 15-cis, dicis isomers, or combinations thereof. [ka]

[0023] The chemical formula for adonixanthine is 3,3'-dihydroxy-β,β-caroten-4-one (C 40 H 54 (03, molecular weight 582.869), and the structural formula of trans adonixanthin is represented by the following formula. In addition, cis adonixanthin includes cis isomers such as 9-cis, 13-cis, and 15-cis, as well as dicis isomers or combinations thereof. [ka]

[0024] Furthermore, according to a preferred embodiment of the present invention, the xanthophyll may be one or more xanthophylls selected from astaxanthin, adonirubin, and adonixanthin.

[0025] Furthermore, xanthophyll in the present invention may be a free form, a fatty acid ester, or a pharmaceutically acceptable salt, and from the viewpoint of absorption, it is preferably a free form. Xanthophyll may also be an optical isomer.

[0026] In the present invention, xanthophyll may also be in the form of a pharmaceutically acceptable salt, and these salts are also included in xanthophyll in the present invention. In the present invention, xanthophyll may also form salts with an acid or a base. In the present invention, a pharmaceutically acceptable salt is not particularly limited as long as it forms a pharmaceutically acceptable salt with xanthophyll. Specifically, examples include, but are not limited to, hydrohalides (e.g., hydrofluorides, hydrochlorides, hydrobroms, hydroiodides, etc.), inorganic salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, bicarbonates, etc.), organic carboxylates (e.g., acetates, oxalates, maleates, tartrates, fumarates, citrates, etc.), organic sulfons (e.g., methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, camphorsulfonates, etc.), amino acid salts (e.g., aspartates, glutamates, etc.), quaternary amine salts, alkali metal salts (e.g., sodium salts, potassium salts, etc.), and alkaline earth metal salts (e.g., magnesium salts, calcium salts, etc.).

[0027] Furthermore, in the present invention, xanthophyll may be a stereoisomer such as an optical isomer. Examples of optical isomers of astaxanthin include at least one selected from the group consisting of the 3S,3'S-isomer, the 3S,3'R-isomer (meso-isomer), and the 3R,3'R-isomer, with the 3S,3'S-isomer being preferred. Examples of optical isomers of adonixanthine include at least one selected from the group consisting of 3S,3'R-isomer, 3S,3'S-isomer, 3R,3'S-isomer, and 3R,3'R-isomer, with the 3S,3'R-isomer being preferred.

[0028] According to one embodiment of the present invention, xanthophyll (preferably trans-xanthophyll) is contained in microorganisms. Specifically, trans-xanthophyll can be produced by a method using microorganisms (for example, microbial fermentation). Such microorganisms include bacteria, algae, and yeasts. Preferred xanthophyll-producing bacteria or yeasts include bacteria or Phaffia yeasts belonging to the genera Paracoccus, Sphingomonas, Brevundimonas, or Erythrobacter, and more preferably bacteria belonging to the genus Paracoccus. Here, preferred species of the genus Paracoccus are Paracoccus carotinifaciens, Paracoccus marcusii, Paracoccus haeundaensis, and Paracoccus zeaxanthinifaciens, with Paracoccus carotinifaciens being more preferred. Specific examples of strains of Paracoccus include Paracoccus carotinifaciens strain E-396 and Paracoccus strain A581-1 (FERM BP-4671), and these mutant strains are also preferably used in the present invention.

[0029] Furthermore, as the xanthophyll-producing bacterium, a bacterium is preferably used in which the base sequence of the DNA corresponding to the 16S ribosomal RNA has high homology (identity) with the base sequence of the E-396 strain. Here, "having high homology" means that the base sequence of the DNA corresponding to the 16S ribosomal RNA of the E-396 strain and the corresponding base sequence of the target bacterium are preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, 98% or more, or 99% or more homologous. The base sequence of the DNA corresponding to the 16S ribosomal RNA of the E-396 strain is listed in the sequence listing of, for example, International Publication No. 2010 / 044469. The base sequence of the DNA corresponding to the 16S ribosomal RNA means the base sequence in which U (uracil) in the base sequence of the 16S ribosomal RNA is replaced with T (thymine).

[0030] According to one embodiment of the present invention, mutant strains with improved xanthophyll productivity can also be used as xanthophyll-producing bacteria. The improved mutant strains can be known strains such as those with high astaxanthin production capacity (for example, the strain described in Japanese Patent Application Publication No. 2001-95500). Furthermore, mutant strains with improved xanthophyll productivity can be obtained by those skilled in the art through mutation treatment and screening based on known techniques. The method of mutation treatment is not particularly limited as long as it induces mutation. For example, chemical methods using mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS), physical methods such as ultraviolet irradiation and X-ray irradiation, and biological methods such as genetic recombination and transposons can be used. The microorganisms subjected to mutation treatment are not particularly limited, but are preferably xanthophyll-producing bacteria. Furthermore, the mutant strains may be those resulting from naturally occurring mutations.

[0031] The method for culturing xanthophyll-producing microorganisms (i.e., the method for producing xanthophyll from xanthophyll-producing microorganisms) and the method for processing said microorganisms are not particularly limited as long as they do not hinder the effects of the present invention. For example, the method for culturing xanthophyll-producing bacteria and the method for processing said bacteria are shown below.

[0032] Method for producing bacterial cells (cultures) For example, xanthophyll-producing bacteria are cultured according to the method of Japanese Patent Publication No. 2007-261972 to produce bacterial cells (culture).

[0033] In the present invention, the xanthophyll production medium used for culturing bacteria is not particularly limited as long as it allows xanthophyll-producing bacteria to grow and produce xanthophyll. Preferably, the xanthophyll production medium contains a carbon source, a nitrogen source, inorganic salts, and vitamins as needed.

[0034] Examples of carbon sources include sugars such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and maltose; organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and pyruvic acid; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and glycerol; and oils and fats such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil, and linseed oil. These carbon sources may be used individually or in combination of two or more. Glucose or sucrose are preferred. The amount added to the culture medium before culturing (initial medium) varies depending on the type of carbon source and can be adjusted as appropriate, but is usually 1g to 100g per liter of medium, preferably 2g to 50g. In addition to adding the carbon source to the initial medium, it is also preferable to add it sequentially or continuously during culturing.

[0035] Examples of inorganic nitrogen sources include ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium phosphate; nitrates such as potassium nitrate; ammonia, and urea. These inorganic nitrogen sources may be used individually or in combination of two or more. The amount added varies depending on the type of inorganic nitrogen source and can be adjusted as appropriate, but is usually 0.1g to 20g per liter of culture medium, preferably 0.2g to 10g.

[0036] Examples of organic nitrogen sources include corn steep liquor (including filtered products), Pharmamedia, soybean meal, soybean flour, peanut meal, distiller's sorbate, dried yeast, and monosodium glutamate. These organic nitrogen sources may be used individually or in combination of two or more. The amount added will vary depending on the type of organic nitrogen source and can be adjusted as appropriate, but is usually 0g to 80g per liter of culture medium, preferably 0g to 40g. Inorganic and organic nitrogen sources are usually added to the initial culture medium, but they may also be supplied sequentially or continuously.

[0037] Examples of inorganic salts include phosphates such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate; magnesium salts such as magnesium sulfate and magnesium chloride; iron salts such as iron sulfate and iron chloride; calcium salts such as calcium chloride and calcium carbonate; sodium salts such as sodium carbonate and sodium chloride; manganese salts such as manganese sulfate; cobalt salts such as cobalt chloride; copper salts such as copper sulfate; zinc salts such as zinc sulfate; molybdenum salts such as sodium molybdate; nickel salts such as nickel sulfate; selenium salts such as sodium selenite; boric acid and potassium iodide. These inorganic salts may be used individually or in combination of two or more. The amount to be added varies depending on the type of inorganic salt and can be adjusted as appropriate, but is usually 0.0001g to 15g per liter of culture medium. Inorganic salts are usually added to the initial culture medium, but may also be added sequentially or continuously.

[0038] Examples of vitamins include cyanocobalamin, riboflavin, pantothenic acid, pyridoxine, thiamine, ascorbic acid, folic acid, niacin, p-aminobenzoic acid, biotin, inositol, and choline. These vitamins may be used individually or in combination of two or more. The amount added varies depending on the type of vitamin and can be adjusted as appropriate, but is usually 0.001 mg to 1000 mg per liter of culture medium, preferably 0.01 mg to 100 mg. Vitamins are usually added to the initial culture medium, but may also be added sequentially or continuously.

[0039] The xanthophyll production medium used in this invention is used for bacterial culture after sterilization. Sterilization can be carried out as appropriate by those skilled in the art. For example, the medium in a suitable container can be heat-sterilized in an autoclave. Alternatively, it may be sterilized by filtration using a sterile filter.

[0040] The pH of the culture medium should be adjusted to, for example, pH 2 to 12, preferably pH 6 to 9.

[0041] In the present invention, xanthophyll-producing bacteria are inoculated into a xanthophyll-producing medium prepared as described above and cultured under predetermined conditions. Inoculation is performed by appropriately increasing the bacterial strain through seed culture using a test tube, flask, or fermenter, and adding the resulting culture to the xanthophyll-producing medium. The medium used for seed culture is not particularly limited as long as it is a medium in which the xanthophyll-producing bacteria can grow well.

[0042] Culturing is carried out in a suitable culture vessel. The culture vessel can be appropriately selected depending on the culture volume, and examples include test tubes, flasks, and fermenters. The culture temperature is 15°C to 80°C, preferably 20°C to 35°C, and more preferably 25°C to 32°C. The culture period is usually 1 to 20 days, preferably 2 to 12 days, and more preferably 3 to 9 days. Culturing is carried out under aerobic conditions. Examples of aerobic conditions include shaking culture or aeration stirring culture, and it is preferable to control the dissolved oxygen concentration within a certain range. The dissolved oxygen concentration can be controlled, for example, by changing the stirring speed, aeration rate, and internal pressure. The dissolved oxygen concentration is preferably controlled to 0.3 ppm to 10 ppm, more preferably 0.5 ppm to 7 ppm, and even more preferably 1 ppm to 5 ppm.

[0043] According to one embodiment of the present invention, after culturing is complete, only the culture medium components are removed from the culture, such as a bacterial culture medium, based on known techniques. The bacterial cells may then be dried using a drum dryer. In addition to a drum dryer, other drying methods such as spray drying, granulation spray drying, and freeze-drying can be used.

[0044] As described above, xanthophyll-producing bacteria can be cultured and dried using a drum dryer, spray drying, granulation spray drying, freeze-drying, etc.

[0045] According to another embodiment of the present invention, a concentrate containing xanthophyll and bacterial cells is separated from the culture obtained by culturing xanthophyll-producing bacteria as described above by centrifugation, filtration, or decantation. The separation step may also be carried out under acidic conditions. Herein, “culture” means any of the following: culture medium, culture supernatant, cultured bacterial cells, culture concentrate obtained from the culture, dried bacterial cells, or lysates of bacterial cells.

[0046] While the culture can be separated directly, it is also preferable to dilute the culture with water before separation to enhance the removal of unwanted components. Water can also be added during operations such as centrifugation, filtration, and decantation. Furthermore, heat sterilization can be performed to kill the cultured microorganisms after cultivation and before separation.

[0047] In the present invention, the method for separating bacterial cells can be a method based on sedimentation or a method based on particle size. Specifically, such a method may involve using centrifugation, filtration, or decantation, either individually or in combination of two or more methods. Alternatively, the same separation method may be repeated two or more times, such as performing centrifugation once and then centrifugating only the supernatant again to recover the xanthophyll remaining in the supernatant. Centrifugation, filtration, or decantation can be carried out appropriately based on known techniques by those skilled in the art.

[0048] The culture concentrate obtained from the culture by the above separation method contains a concentration of xanthophyll and bacterial cells. It is also preferable to adjust the separation rate, separation strength, etc., as appropriate so that the culture concentrate has a viscosity and water content suitable for the next step.

[0049] The microorganisms or microbial products obtained as described above contain one or more xanthophylls, such as astaxanthin, adonirubin, and adonixanthin.

[0050] Microorganisms containing trans xanthophyll are exposed to supercritical CO2. 2 and a step of contacting astaxanthin with a fluid containing an organic solvent in which the solubility of astaxanthin at 25°C is 0.001 g / L to 0.4 g / L, or with a fluid containing an alcohol having 1 to 6 carbon atoms. The present invention provides a method for isomerizing trans xanthophyll to cis xanthophyll, comprising the steps of contacting a microorganism containing trans xanthophyll with a fluid containing supercritical CO2 and an organic solvent (hereinafter also referred to as a specific organic solvent) having a solubility of astaxanthin of 0.001 g / L to 0.4 g / L at 25°C, or a fluid containing an alcohol having 1 to 6 carbon atoms, under temperature conditions of 70 to 160°C (hereinafter also referred to as a contact step). Hereinafter, the specific organic solvent and the alcohol having 1 to 6 carbon atoms are collectively referred to as the co-medium.

[0051] The isomerization method of the present invention will be explained with reference to Figure 1. Figure 1 is a schematic diagram of a supercritical extraction apparatus used in the isomerization method of the present invention. In Figure 1, the supercritical extraction apparatus (100) comprises a condenser-equipped booster pump (1) with a cooler for pressurizing and supplying CO2 (2), a booster pump (13) for supplying a co-medium (3), an extraction tank (4) comprising a heater (6) for heating the CO2 and co-medium, and a container (5) containing microorganisms containing trans-type xanthophyll, and a separation unit (12) for returning the fluid to atmospheric pressure and separating the fluid (11) from the cis-type xanthophyll-containing composition (10). In the extraction tank (4), a fluid containing supercritical CO2 and co-medium is introduced into the container (5) containing microorganisms containing trans-type xanthophyll, bringing the microorganisms and the fluid into contact to extract the cis-type xanthophyll-containing composition. Furthermore, in Figure 1, the supercritical extraction apparatus (100) is equipped with a back pressure valve (8) for controlling the pressure in the extraction tank (4), a pressure gauge (7) for checking the pressure in the extraction tank (4), a heater (14) for preventing the back pressure valve (8) from freezing due to the heat of vaporization of carbon dioxide, and a flow meter (9) for checking the flow rate of carbon dioxide. In the isomerization method of the present invention, cis-type xanthophyll can be efficiently recovered by replacing the microorganisms used as raw materials. The replacement may consist of replacing only the microorganisms, or the entire container (5) containing the microorganisms may be replaced. In addition, as long as xanthophyll is present in the microorganisms used as raw materials, cis-type xanthophyll can be continuously recovered in the separation unit. Furthermore, the supercritical extraction apparatus is not limited to the configuration specified in Figure 1, and may be modified as appropriate based on common technical knowledge in the art, as long as it achieves the effects of the invention.

[0052] (fluid) The fluid contains supercritical CO2 and a co-medium. The fluid may contain additives (e.g., antioxidants, vegetable oils, etc.) or enhancers (e.g., water, etc.), as long as they do not produce the effects of the present invention. Preferably, the fluid consists of supercritical CO2 and a co-medium.

[0053] "Supercritical CO2" refers to carbon dioxide that exists under conditions of a critical temperature of 31.1°C or higher and a critical pressure of 7.38 MPa or higher.

[0054] "Specific organic solvents" refer to organic solvents in which the solubility of astaxanthin at 25°C is 0.001 g / L to 0.4 g / L. The solubility of astaxanthin in specific organic solvents at 25°C is preferably 0.01 g / L to 0.38 g / L, more preferably 0.015 g / L to 0.36 g / L, and even more preferably 0.018 g / L to 0.2 g / L. Specific organic solvents include, for example, carboxylic acids such as acetic acid (solubility 0.36 g / L) and formic acid (solubility 0.29 g / L); ketones such as acetone (solubility 0.16 g / L), ethyl methyl ketone (solubility 0.26 g / L), and methyl isobutyl ketone (solubility 0.18 g / L); esters such as methyl acetate (solubility 0.21 g / L), ethyl acetate (solubility 0.20 g / L), and butyl acetate (solubility 0.18 g / L); aromatic hydrocarbons such as toluene (solubility 0.30 g / L) and p-xylene (solubility 0.15 g / L); alcohols such as methanol (solubility 0.023 g / L), ethanol (solubility 0.018 g / L), and isopropyl alcohol (solubility 0.013 g / L); and cyclohexane (solubility 0.008 g / L), hexane (solubility Examples include aliphatic hydrocarbons such as 0.002 g / L. Among these, specific organic solvents are preferably acetic acid, formic acid, acetone, ethyl methyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, butyl acetate, toluene, p-xylene, methanol, ethanol, and isopropyl alcohol; more preferably acetic acid, formic acid, acetone, ethyl methyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, butyl acetate, toluene, p-xylene, methanol, and ethanol; and even more preferably acetone, methyl isobutyl ketone, ethyl acetate, butyl acetate, p-xylene, methanol, and ethanol. Furthermore, certain organic solvents may be used individually or in combination of two or more.

[0055] Alcohols having 1 to 6 carbon atoms include linear, branched, or cyclic alcohols. More specifically, examples include methanol, ethanol, n-propyl alcohol, i-propyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, n-pentyl alcohol, and n-hexyl alcohol. Among alcohols having 1 to 6 carbon atoms, alcohols having 1 to 3 carbon atoms are preferred, and ethanol is more preferred. Alcohols with 1 to 6 carbon atoms may be used individually or in combination of two or more.

[0056] The concentration of the co-media is not particularly limited as long as it does not hinder the effects of the present invention, but for example, it is 5% to 90% by mass relative to supercritical CO2, more preferably 10% to 75% by mass, and even more preferably 15% to 50% by mass. The presence of the co-media in the fluid improves the solubility of xanthophyll and can improve the yield of the cis isomer.

[0057] While there are no particular limitations on the antioxidants used, those used as food additives are preferred due to their ease of application to humans and animals. The type of antioxidant can be appropriately selected depending on the type of xanthophyll. Examples of antioxidants include ascorbic acid (VC), α-tocopherol, propyl gallate, butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), ascorbyl palmitate (PAVC), coenzyme Q10, resveratrol, and curcumin. Preferably, the antioxidants are ascorbic acid and α-tocopherol. These antioxidants may be used individually or in combination of two or more.

[0058] The concentration of the antioxidant in the above fluid is not particularly limited as long as it does not hinder the effects of the present invention, but is preferably 0.01 to 20% by mass, more preferably 0.1 to 10% by mass, and even more preferably 0.5 to 5% by mass.

[0059] The vegetable oil is not particularly limited, and its type can be appropriately selected depending on the type of xanthophyll. Examples of vegetable oils include soybean oil, mustard oil, hemp seed oil, sesame oil, safflower oil, rice oil, argan oil, olive oil, sunflower oil, macadamia oil, and palm oil. The vegetable oils are preferably soybean oil and mustard oil. These vegetable oils may be used individually or in combination of two or more types.

[0060] The concentration of vegetable oil in the above fluid is not particularly limited as long as it does not hinder the effects of the present invention, but is preferably 0.01 to 20% by mass, more preferably 0.1 to 10% by mass, and even more preferably 0.5 to 5% by mass.

[0061] The heating temperature in the contact step is 70 to 160°C. From the viewpoint of promoting the cis isomerization reaction, it is preferably 80°C or higher, more preferably exceeding 90°C, and even more preferably 100°C or higher. From the viewpoint of suppressing the decomposition of xanthophyll, the heating temperature in the contact step is preferably 150°C or lower, more preferably 140°C or lower, even more preferably 130°C or lower, and even more preferably 120°C or lower.

[0062] The pressure in the contact step is not particularly limited within the range in which CO2 becomes supercritical CO2. From the viewpoint of promoting the cis isomerization reaction, the pressure in the contact step is preferably 50 MPa or less, and more preferably 45 MPa or less. From the viewpoint of extracting xanthophyll from microorganisms, the pressure in the contact step is preferably 20 MPa or more, and more preferably 25 MPa or more.

[0063] The contact time in the contact process is not limited as long as the effects of the present invention are achieved, but examples include 1 minute to 240 minutes, preferably 1 minute to 180 minutes, more preferably 3 minutes to 150 minutes, and even more preferably 5 minutes to 120 minutes. The contact time may be appropriately set depending on the heating temperature. For example, when the heating temperature is 80°C to 160°C, the contact time is preferably 1 minute to 180 minutes, for example when the heating temperature is 100°C to 160°C, the contact time is preferably 3 minutes to 150 minutes, and for example when the heating temperature is 120°C to 140°C, the contact time is preferably 5 minutes to 120 minutes.

[0064] According to a preferred embodiment of the isomerization method of the present invention, the conditions for the contact step are as follows: the fluid consists of supercritical CO2 and ethanol; the heating temperature is 90°C to 130°C; the pressure is 25 MPa to 45 MPa; the contact time is 5 minutes to 120 minutes; and the ethanol concentration is 15% to 50% by mass.

[0065] According to one embodiment of the present invention, the isomerization method of the present invention may include a cooling step (also called a cooling step) after the contact step. The cooling temperature of the cooling step should be lower than the lower limit of the heating temperature of the contact step, and from the viewpoint of suppressing the decomposition of xanthophyll, for example, it should be 70°C or lower, preferably 60°C or lower, and more preferably 40°C or lower. The lower limit of the cooling temperature of the cooling step is usually room temperature (for example, 25°C). Cooling can be performed, for example, by cooling the container constituting the separation unit.

[0066] According to another embodiment of the isomerization method of the present invention, the step of removing the co-media from the cis-type xanthophyll-containing composition may further include a step of removing the co-media after the cooling step, which may be, for example, a step of evaporating the co-media at 25-30°C and low pressure.

[0067] Composition containing cis-type xanthophyll According to another aspect of the present invention, a cis-xanthophyll-containing composition is provided. Such a composition contains at least cis-xanthophyll. At least a portion of the cis-xanthophyll is obtained by cis-isomerizing trans-xanthophyll contained in a microorganism. The above composition may contain any components other than cis-xanthophyll and microorganism-derived components. Examples of such optional components include the above-mentioned antioxidants, vegetable oils, and residual components of fluids. Here, the composition is not the microorganism itself, but rather a composition in which the cis-xanthophyll content has been increased by the above-mentioned isomerization method. The cis-xanthophyll-containing composition may also be referred to as a processed product of a microorganism containing cis-xanthophyll, a cis-isomerized product, or a cis-isomerized product.

[0068] In a preferred embodiment, the cis-xanthophyll-containing composition of the present invention includes a cis-xanthophyll obtained by the isomerization method described above as the cis-xanthophyll. The cis-xanthophyll is preferably cis-adonirubin, cis-adonixanthin, cis-astaxanthin, cis-zeaxanthin, or cis-β-cryptoxanthin, and more preferably cis-astaxanthin, cis-adonirubin, or cis-adonixanthin. These xanthophylls may be present individually or in combination of two or more.

[0069] (Cis-isomer ratio) The cis-isomer ratio in the cis-xanthophyll-containing composition of the present invention refers to the proportion of cis-xanthophyll to the total amount of cis-xanthophyll and trans-xanthophyll in the cis-xanthophyll-containing composition (hereinafter also referred to as the total amount of xanthophyll). More specifically, the cis-isomer ratio can be determined by HPLC analysis of the cis-xanthophyll-containing composition using the following formula. Here, the definition of the total amount of xanthophyll also applies to each xanthophyll such as astaxanthin, adonirubin, and adonixanthin.

number

[0070] In the cis-type xanthophyll-containing composition of the present invention, the cis-isomer ratio (area %) when the total amount of xanthophyll is 100 area %, is not particularly limited, but for example, it is 10 area % or more, preferably 20 area % or more, more preferably 30 area % or more, and even more preferably 40 area % or more, 50 area % or more, or 60 area % or more. The upper limit of the cis-isomer ratio is not particularly limited, but for example, it is 90 area % or less, preferably 80 area % or less, and more preferably 70 area % or less.

[0071] In the cis-xanthophyll-containing composition of the present invention, the cis-isomer ratio and xanthophyll recovery rate can be measured by HPLC (high-performance liquid chromatography) using a normal-phase column or a reversed-phase column, preferably a normal-phase column. Quantification is performed based on the peak area of ​​each xanthophyll isomer peak in the chromatogram. If the peaks are separated, the peak area is the area obtained by connecting the peak start and end points; if the peaks overlap, the peak area is the area obtained by vertically splitting from the minimum value between the peaks. Such measurements can be easily performed using commercially available HPLC instruments (e.g., Shimadzu Corporation) and columns (e.g., Luna, 5 μm, Silica(2), 100 Å (150 mm × φ4.6 mm) (Phenomenex)). The above measurements can be performed under the following conditions: Instrument: High-performance liquid chromatograph Prominence system (SPD-M20A, Shimadzu Corporation), Column: Two Luna, 5 μm, Silica(2), 100 Å (150 mm × φ4.6 mm) (Phenomenex) columns linked together, Mobile phase: Hexane / ethyl acetate / acetone (70:20:10, v / v / v), Column temperature: 40°C, Flow rate: 1.2 mL / min, Detection wavelength: 470 nm.

[0072] HPLC samples can be prepared as follows. First, the cis-xanthophyll-containing composition is suspended in acetone and sonicated at 10°C for 15 minutes, then filtered through a filter (preferably a filter with a pore size of 0.22 μm, more preferably a PTFE filter with a pore size of 0.22 μm). Next, the solvent is removed from the obtained filtrate by evaporation under reduced pressure at 35°C, then dissolved in ethyl acetate / hexane (volume ratio 70:30), filtered again through the same filter, and an HPLC sample is obtained. The method for preparing HPLC samples from cultures is the same as the method for preparing HPLC samples from the cis-xanthophyll-containing composition described above.

[0073] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-astaxanthin. The ratio of the cis-isomer of astaxanthin in the cis-xanthophyll-containing composition (= (sum of peak areas of cis-astaxanthin / sum of peak areas of cis-astaxanthin and trans-astaxanthin) × 100) is, for example, 22 area% or more, preferably 30 area% or more, more preferably 40 area% or more, and even more preferably 51 area% or more. The upper limit of the ratio of the cis-isomer of astaxanthin is not particularly limited, but is preferably 80 area% or less, more preferably 70 area% or less, and even more preferably 60 area% or less. The above astaxanthin is preferably endogenous. Here, endogenous xanthophyll and endogenous cis-xanthophyll refer to endogenous xanthophyll derived from microorganisms and include cis-xanthophyll isomerized by the isomerization method of the present invention. The definitions of endogenous xanthophyll and endogenous cis-type xanthophyll also apply to other xanthophylls such as astaxanthin, adonirubin, and adonixanthin.

[0074] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-adonirubin. The ratio of the cis-isomer of adonirubin in the cis-xanthophyll-containing composition (= (sum of peak areas of cis-adonirubin / sum of peak areas of cis-adonirubin and trans-adonirubin) × 100) is, for example, 24 area% or more, preferably 30 area% or more, more preferably 40 area% or more, and even more preferably 57 area% or more. The upper limit of the ratio of the cis-isomer of adonirubin is not particularly limited, but is preferably 80 area% or less, and more preferably 70 area% or less. The adonirubin is preferably endogenous.

[0075] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-adonixanthin. The cis-isomer ratio of adonixanthin in the cis-xanthophyll-containing composition (= (sum of peak areas of cis-adonixanthin / sum of peak areas of cis-adonixanthin and trans-adonixanthin) × 100) is, for example, 22 area% or more, preferably 30 area% or more, more preferably 40 area% or more, and even more preferably 47 area% or more. The upper limit of the cis-isomer ratio of adonixanthin is not particularly limited, but is preferably 80 area% or less, more preferably 70 area% or less, and even more preferably 60 area% or less. The adonixanthin is preferably endogenous.

[0076] (Xanthophyll recovery rate) In the cis-type xanthophyll-containing composition of the present invention, the xanthophyll recovery rate refers to the ratio of the xanthophyll content after isomerization treatment (specifically, after the contact step) to the xanthophyll content before isomerization treatment (specifically, before the contact step). The xanthophyll recovery rate can be determined by HPLC analysis using the following formula.

number

[0077] The xanthophyll recovery rate (hereinafter also referred to as the recovery rate) in the cis-type xanthophyll-containing composition of the present invention is not particularly limited, but for example, it is 10 area% or more, preferably 20 area% or more, more preferably 30 area% or more, and even more preferably 40 area% or more. The upper limit of the xanthophyll recovery rate is not particularly limited, but for example, it may be 90 area% or less, and may be 80 area% or less. The xanthophyll is preferably endogenous.

[0078] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-astaxanthin. The astaxanthin recovery rate in the above cis-xanthophyll-containing composition (= (sum of peak areas of all astaxanthin after isomerization treatment / sum of peak areas of all astaxanthin before isomerization treatment) × 100) is, for example, 5 area% or more. Such an astaxanthin recovery rate is preferably 15 area% or more, more preferably 25 area% or more, and even more preferably 35 area% or more. The upper limit of the astaxanthin recovery rate is not particularly limited, but for example, it is 80 area% or less, preferably 70 area% or less, and more preferably 60 area% or less. The above astaxanthin is preferably endogenous.

[0079] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-adonirubin. The adonirubin recovery rate in the above cis-xanthophyll-containing composition (= (sum of peak areas of all adonirubin after isomerization treatment / sum of peak areas of all adonirubin before isomerization treatment) × 100) is, for example, 10 area% or more. Such an adonirubin recovery rate is preferably 20 area% or more, more preferably 30 area% or more, and even more preferably 40 area% or more. The upper limit of the adonirubin recovery rate is not particularly limited, but for example, it is 80 area% or less, and preferably 70 area% or less. The above adonirubin is preferably endogenous.

[0080] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-adonixanthin. The adonixanthin recovery rate in the above cis-xanthophyll-containing composition (= (sum of peak areas of all adonixanthin after isomerization treatment / sum of peak areas of all adonixanthin before isomerization treatment) × 100) is, for example, 10 area% or more. Such an adonixanthin recovery rate is preferably 20 area% or more, more preferably 30 area% or more, and even more preferably 40 area% or more. The upper limit of the adonixanthin recovery rate is not particularly limited, but for example, it is 80 area% or less, and preferably 70 area% or less. The above adonixanthin is preferably endogenous.

[0081] (Yield of cis-type xanthophyll) The yield of cis-type xanthophyll (also called the cis-isomer yield) in the cis-type xanthophyll-containing composition of the present invention is calculated by multiplying the above-mentioned cis-isomer ratio by the xanthophyll recovery rate. The yield of cis-type xanthophyll is not particularly limited, but for example, it may be 10% or more, preferably 20% or more, more preferably 30% or more, and even more preferably 40% or more. The upper limit of the yield of cis-type xanthophyll is not particularly limited, but for example, it may be 90% or less, and may be 80% or less.

[0082] According to one embodiment of the present invention, a cis-xanthophyll-containing composition contains cis-astaxanthin. The yield of cis-astaxanthin in the above cis-xanthophyll-containing composition is calculated by multiplying the ratio of the cis-isomer of the astaxanthin by the astaxanthin recovery rate. The yield of such cis-astaxanthin is not particularly limited, but for example, it may be 4% or more, preferably 10% or more, more preferably 20% or more, and even more preferably 30% or more. The upper limit of the yield of cis-astaxanthin is not particularly limited, but for example, it may be 50% or less, and may be 40% or less.

[0083] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-adonirubin. The yield of cis-adonirubin in the cis-xanthophyll-containing composition is calculated by multiplying the cis-isomer ratio of adonirubin by the adonirubin recovery rate. The yield of cis-adonirubin is not particularly limited, but for example, it may be 6% or more, preferably 10% or more, more preferably 20% or more, and even more preferably 30% or more. The upper limit of the yield of cis-adonirubin is not particularly limited, but for example, it may be 50% or less, and may be 40% or less.

[0084] According to one embodiment of the present invention, the cis-xanthophyll-containing composition contains cis-adonixanthin. The yield of cis-adonixanthin in the cis-xanthophyll-containing composition is calculated by multiplying the cis-isomer ratio of the adonixanthin by the adonixanthin recovery rate. The yield of cis-adonixanthin is not particularly limited, but for example, it may be 7% or more, preferably 10% or more, more preferably 20% or more, and even more preferably 30% or more. The upper limit of the yield of cis-adonixanthin is not particularly limited, but for example, it may be 50% or less, and may be 40% or less.

[0085] According to a preferred embodiment of the present invention, all xanthophylls in the cis-xanthophyll-containing composition are endogenous. Furthermore, according to another preferred embodiment of the present invention, the xanthophylls in the cis-xanthophyll-containing composition are free.

[0086] The cis-xanthophyll-containing composition of the present invention is obtained by contacting a microorganism containing the trans-xanthophyll with a fluid containing supercritical CO2 and a co-medium under temperature conditions of 70°C to 160°C, and then drying, grinding, etc., as necessary. Therefore, the cis-xanthophyll-containing composition can be a dried product or a ground product, and is preferably a dried product.

[0087] According to another aspect of the present invention, a cis-xanthophyll-containing composition derived from a microorganism is provided. Such a cis-xanthophyll-containing composition is preferably a processed product of a microorganism containing cis-xanthophyll.

[0088] According to one embodiment of the cis-xanthophyll-containing composition of the present invention, the composition may contain any component other than the microbial-derived component along with the microbial-derived cis-xanthophyll. Examples of such optional components include the antioxidant, vegetable oil, and residual components of the fluid. Furthermore, in addition to the above optional components, the composition may also be provided with orally acceptable or pharmaceutically acceptable additives as desired. Examples of such additives include solvents, solubilizers, solvents, lubricants, emulsifiers, isotonic agents, stabilizers, preservatives, antiseptics, surfactants, gelling agents, modifiers, chelating agents, pH adjusters, buffers, excipients, thickeners, colorants, fragrances, sweeteners, or flavorings.

[0089] In a preferred embodiment, the cis-xanthophyll-containing composition of the present invention may contain natural products, and more preferably, the cis-xanthophyll-containing composition may consist solely of natural products. Here, "natural products" refers to products derived solely from microorganisms and that do not contain additives such as preservatives (i.e., additive-free products).

[0090] The cis-type xanthophyll-containing composition of the present invention is not particularly limited, but can be used as animal feed (preferably color-enhancing feed), food or beverages (preferably health foods), food additives, pharmaceuticals, quasi-drugs, or cosmetics, or as a raw material for such animal feeds. The cis-type xanthophyll-containing composition undergoes a cis-isomerization reaction in a contact step. Examples of such fluids include fluids containing supercritical CO2 and a co-medium, and considering safety for animals such as humans, a fluid consisting of supercritical CO2 and ethanol is preferably used.

[0091] According to one embodiment of the present invention, the cis-type xanthophyll-containing composition of the present invention can be produced by a manufacturing method that includes the step of contacting a microorganism containing trans-type xanthophyll with a fluid containing supercritical CO2 and a co-medium under temperature conditions of 70°C to 160°C.

[0092] The above embodiments of the manufacturing method can be carried out in accordance with the description of the method for isomerizing trans xanthophyll to cis xanthophyll or the description of the cis xanthophyll-containing composition of the present invention.

[0093] The method of ingesting or administering the cis-type xanthophyll-containing composition of the present invention is not particularly limited, but includes infusion, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, etc., as well as oral ingestion or administration via mucous membrane, transdermal, nasal cavity, oral cavity, abdominal cavity, etc., and is preferably oral ingestion or oral administration.

[0094] The intake or administration amount of the cis-xanthophyll-containing composition of the present invention is not particularly limited and can be determined depending on the formulation of the cis-xanthophyll-containing composition, the type and purity of the xanthophyll, the type of subject, the age or weight of the subject, the symptoms, the time of intake or administration, the form of the cis-xanthophyll-containing composition, the method of intake or administration, and any combination of xanthophyll other than the xanthophyll of the present invention or any other drug. The cis-xanthophyll-containing composition of the present invention is preferably composed in a form that provides an effective amount for color enhancement, improved reproduction rate, nutritional enhancement of egg yolk, improved heat stress tolerance, or improved immunity. For example, when the cis-xanthophyll-containing composition of the present invention is taken or administered orally, the cis-xanthophyll-containing composition can be prepared or formulated so that the amount of xanthophyll is, for example, 0.01 to 1000 mg / kg of body weight, preferably 0.05 to 500 mg / kg of body weight.

[0095] Furthermore, the daily intake or dosage of the cis-xanthophyll-containing composition of the present invention is appropriately selected according to the formulation of the cis-xanthophyll-containing composition, similar to the intake or dosage of the cis-xanthophyll-containing composition described above. The daily intake or dosage of the cis-xanthophyll-containing composition of the present invention may be administered to the subject once or multiple times, but it is preferable to administer it to the subject 1 to 5 times. Therefore, the number of times the cis-xanthophyll-containing composition of the present invention is taken or administered per day is 1 to 5 times, preferably 1 to 3 times, and more preferably once a day.

[0096] According to one embodiment of the present invention, the target to which the cis-xanthophyll-containing composition of the present invention is applied is not particularly limited, as long as it does not hinder the effects of the present invention, but is preferably fish, crustaceans, mammals, and birds, and more preferably salmon, trout, sea bream, crabs, shrimp, krill, ruminants such as cattle and sheep, primates such as humans, and poultry such as dogs, cats, pigs, and chickens. The target may be a healthy person (healthy animal) or a patient (patient animal).

[0097] Another aspect of the present invention provides a method for enhancing the color of a target, improving the reproduction rate of a target, enhancing the nutritional value of the egg yolk of a target, improving the heat stress tolerance of a target, or improving the immunity of a target, the method comprising administering or ingesting an effective amount of a cis-xanthophyll-containing composition to a target in need. Here, "effective amount" can be set in the same way as the content of the cis-xanthophyll-containing composition in a daily intake unit. Here, "enhancing the color" includes making the color of fish meat, egg yolk, etc., a color that matches consumer preferences.

[0098] Furthermore, according to another aspect of the present invention, the use of a cis-xanthophyll-containing composition or a cis-xanthophyll composition derived from microorganisms is provided for color enhancement, improved reproduction rate, enhanced nutrition of egg yolk, improved heat stress tolerance, or improved immunity.

[0099] Furthermore, according to another aspect of the present invention, the use of a cis-xanthophyll-containing composition is provided as a composition for enhancing color, improving reproduction rates, strengthening the nutritional value of egg yolks, improving heat stress tolerance, or improving immunity.

[0100] Furthermore, according to another aspect of the present invention, the use of a cis-xanthophyll-containing composition in the production of a composition for color enhancement, improved reproduction rate, enhanced nutrition of egg yolk, improved heat stress tolerance, or improved immunity is provided.

[0101] Furthermore, according to another aspect of the present invention, a cis-xanthophyll-containing composition is provided for color enhancement, improved reproduction rate, enhanced nutrition of egg yolk, improved heat stress tolerance, or improved immunity.

[0102] The above methods, uses, and embodiments of the cis-type xanthophyll-containing composition for color enhancement can be carried out in accordance with the description of the method for isomerizing trans-type xanthophyll to cis-type xanthophyll or the cis-type xanthophyll-containing composition of the present invention. [Examples]

[0103] The present invention will be described in more detail below with reference to preparation and test examples, but the technical scope of the present invention is not limited to these examples. Unless otherwise specified, the units and measurement methods described herein are in accordance with JIS standards.

[0104] Preparation Example 1: Preparation of Paracoccus Dried Powder Paracoccus carotinifaciens strain E-396 (FERM EP-4283) was cultured according to the method described in Example 1 of Japanese Patent Publication No. 2007-261972. Specifically, 10 ml of a culture medium consisting of 2 g / L glucose, 3 g / L meat extract, 10 g / L peptone, and 5 g / L sodium chloride was placed in a test tube and steam-sterilized at 121°C for 15 minutes. Strain E-396 (FERM BP-4283) was inoculated into this medium using a platinum loop and cultured at 30°C for 6 days with reciprocating shaking at 300 rpm. After centrifuging the culture solution, it was freeze-dried to obtain dried Paracoccus powder.

[0105] Test Example 1: Isomerization reaction of trans xanthophyll (supercritical CO2 2 Temperature study in contact with a fluid consisting of ethanol. A supercritical extraction apparatus was prepared, comprising a booster pump with a cooler for pressurizing and supplying CO2, a booster pump for supplying ethanol, a heater for heating CO2 and alcohol, an extraction tank for extracting a cis-type xanthophyll-containing composition by contacting microorganisms containing trans-type xanthophyll with a fluid consisting of supercritical CO2 and ethanol, and a separation unit for returning the fluid to atmospheric pressure and separating the fluid from the cis-type xanthophyll-containing composition. One g of the dried Paracoccus powder obtained in Preparation Example 1 was placed in an extraction tank and brought into contact with a fluid consisting of supercritical CO2 and ethanol under heating conditions to carry out the isomerization reaction of trans xanthophyll. The pressure was 30 MPa, the heating temperature was varied between 60°C and 140°C, and the reaction time was varied between 30 minutes and 240 minutes. Subsequently, the cis xanthophyll-containing composition was obtained by separating the fluid in a separation unit. The flow rate of ethanol was 0.5 mL / min, and the flow rate of CO2 was 3.0 mL / min.

[0106] The obtained composition was suspended in acetone and sonicated at 10°C for 15 minutes, then filtered through a PTFE filter (Osaka Chemical Co., Ltd., pore size 0.22 μm). The obtained filtrate was evaporated under reduced pressure at 35°C to remove the solvent, then dissolved in ethyl acetate / hexane (volume ratio 70:30), and filtered again through a PTFE filter to obtain a sample for HPLC (hereinafter also referred to as "sample"). The obtained HPLC sample was analyzed by HPLC under the following analytical conditions, and the xanthophyll recovery rate (also referred to as "recovery rate"), cis isomer ratio (also referred to as "cis ratio"), and cis isomer yield (also referred to as "cis yield") of astaxanthin, adonirubin, and adonixanthin were calculated based on the peak area in the obtained chromatogram.

[0107] The above HPLC was performed under the following conditions. Equipment: High-performance liquid chromatograph Prominence system (SPD-M20A, manufactured by Shimadzu Corporation) Two columns of Luna, 5μm, Silica(2), 100Å (150mm × φ4.6mm) (manufactured by Phenomenex) were used in conjunction. Mobile phase: Hexane / ethyl acetate / acetone (70:20:10, v / v / v) Column temperature: 40℃ Flow rate: 1.2mL / min Detection wavelength: 470nm

[0108] The results are shown in Table 1. The values ​​for each sample are shown as the mean and standard deviation (n=3). Furthermore, analysis of the dried Paracoccus powder by HPLC under the above analytical conditions before contact with a fluid consisting of supercritical CO2 and ethanol revealed that the trans-form content of astaxanthin, adonirubin, and adonixanthin was 95.5 area%, 90.7 area%, and 100.0 area%, respectively.

[0109] [Table 1-1] [Table 1-2] [Table 1-3]

[0110] As is clear from Table 1, an improvement in the cis isomer ratio was observed with increasing reaction temperature. Here, from the viewpoint of xanthophyll recovery rate and cis isomer yield, a reaction temperature in the range of 80°C to 140°C was preferred, and a range of 100°C to 140°C was considered more preferred.

[0111] Test Example 2: Investigation of pressure in the isomerization reaction of trans xanthophyll The contact process conditions were set to pressures of 20 MPa, 30 MPa, and 40 MPa, and the heating temperatures were set to 80°C and 120°C for each pressure, but otherwise the same tests as in Test Example 1 were conducted, and the recovery rates, cis-isomer ratio, and cis-isomer yield of astaxanthin, adonirubin, and adonixanthin were calculated. The results are shown in Table 2. The values ​​for each sample are shown as the mean and standard deviation (n=3).

[0112] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0113] Table 2 shows that higher reaction pressures tended to improve the recovery rate of xanthophyll. Therefore, from the viewpoint of the yield of the cis isomer of xanthophyll, a reaction pressure in the range of 30 MPa to 40 MPa was considered preferable.

[0114] Test Example 3: Investigation of ethanol supply amount in the isomerization reaction of trans xanthophyll Under contact process conditions of 120°C and 30 MPa pressure, the ethanol supply rate was changed to 0 mL / min, 0.5 mL / min, and 1.0 mL / min. Under contact process conditions of 80°C and 30 MPa pressure, the ethanol supply rate was changed to 0 mL / min and 0.5 mL / min. The same procedure as in Test Example 1 was performed except for these changes, and the recovery rates, cis-isomer ratios, and cis-isomer yields of astaxanthin, adonirubin, and adonixanthin were calculated. The results are shown in Table 3. The values ​​for each sample are shown as the mean and standard deviation (n=3).

[0115] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0116] As is clear from Table 3, even without the addition of ethanol, the cis isomer ratio was high, but the recovery rate decreased significantly, resulting in a decline in cis isomer yield. Furthermore, while the cis isomer ratio decreased slightly with the addition of ethanol, the cis isomer yield improved significantly. Therefore, it is thought that the isomerization of trans xanthophylls occurs during extraction with a fluid containing supercritical CO2, but an alcoholic solvent such as ethanol is considered necessary for the extraction of xanthophylls. [Explanation of Symbols]

[0117] 1: Booster pump with condenser, 2: Carbon dioxide, 3: Co-medium, 4: Extraction tank, 5: Container, 6, 14: Heater, 7: Pressure gauge, 8: Back pressure valve, 9: Carbon dioxide flow meter, 10: Cis-type xanthophyll-containing composition, 11: Fluid, 12: Separation unit, 13: Booster pump, 100: Supercritical fluid extraction apparatus.

Claims

1. Microorganisms containing trans xanthophyll are subjected to supercritical CO2 under temperature conditions of 70 to 160°C. ２ A method comprising a contact step of contacting a fluid containing an organic solvent in which the solubility of astaxanthin at 25°C is 0.001 g / L to 0.4 g / L, wherein the trans xanthophyll isomerized to cis xanthophyll in the contact step.

2. Microorganisms containing trans xanthophyll are subjected to supercritical CO2 under temperature conditions of 70 to 160°C. ２ A method comprising a contact step of contacting a fluid containing an alcohol having 1 to 6 carbon atoms, wherein the trans xanthophyll isomerized to a cis xanthophyll in the contact step.

3. The method according to claim 2, wherein the alcohol is ethanol.

4. The method according to any one of claims 1 to 3, wherein the temperature condition is 80°C to 140°C.

5. The method according to any one of claims 1 to 4, wherein the pressure condition in the contact step is 20 MPa or more.

6. The concentration of the organic solvent or the alcohol is the supercritical CO ２ The method according to any one of claims 1 to 5, wherein the amount is 15% by mass to 50% by mass.

7. The method according to any one of claims 1 to 6, wherein the microorganism is Paracoccus carotinifaciens.

8. The method according to any one of claims 1 to 7, wherein the trans xanthophyll is at least one selected from the group consisting of trans astaxanthin, trans adonirubin, trans adonixanthin, trans zeaxanthin, and trans β-cryptoxanthin.

9. Microorganisms containing trans xanthophyll are subjected to supercritical CO2 under temperature conditions of 70-160°C. ２ A method for producing a cis-type xanthophyll-containing composition, comprising a contact step of contacting the fluid with an organic solvent having a solubility of astaxanthin of 0.001 g / L to 0.4 g / L at 25°C, wherein the trans-type xanthophyll isomerized to cis-type xanthophyll in the contact step.

10. Microorganisms containing trans xanthophyll are subjected to supercritical CO2 under temperature conditions of 70-160°C. ２ A method for producing a cis-type xanthophyll-containing composition, comprising a contact step of contacting the composition with a fluid containing an alcohol having 1 to 6 carbon atoms, wherein the trans-type xanthophyll isomerized to a cis-type xanthophyll in the contact step.

11. The method according to claim 9 or 10, wherein the composition comprises at least one selected from the group consisting of cis-astaxanthin, cis-adonilbin, and cis-adonixanthin.

12. The method according to claim 11, wherein the proportion of the cis isomer of astaxanthin in the composition is 22 area percent or more, the proportion of the cis isomer of adonilubin is 24 area percent or more, or the proportion of the cis isomer of adonixanthin is 22 area percent or more.

13. The method according to any one of claims 9 to 12, wherein the microorganism is Paracoccus carotinifaciens.

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