Method for isomerizing trans-carotenoids to cis-carotenoids

Abstract

To provide novel methods for isomerizing trans carotenoids to cis carotenoids.SOLUTION: A method for isomerizing a trans carotenoid to a cis carotenoid is used, the method comprising treating a microorganism comprising a trans carotenoid in a subcritical fluid, and the fluid containing water.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for isomerizing trans-carotenoids to cis-carotenoids, and more particularly to a method for isomerizing trans-carotenoids to cis-carotenoids, comprising a step of treating a microorganism containing the trans-carotenoid in a subcritical fluid, the fluid containing water. [Background technology]

[0002] Carotenoids are natural pigments that are widely found in nature, and known types of carotenoids include astaxanthin, adonirubin, adonixanthin, zeaxanthin, and β-cryptoxanthin. Astaxanthin, adonirubin, adonixanthin, and the like are known to have various effects, including an anti-anxiety physiological effect (Patent Document 1), and are expected to be used in foods, pharmaceuticals, and the like.

[0003] Astaxanthin, adonirubin, adonixanthin, and the like are widely distributed in animals, plants, and microorganisms. For example, astaxanthin is widely distributed in nature, such as fish such as salmon, trout, and red sea bream, and crustaceans such as crabs, shrimp, and krill. It is also produced by microorganisms such as bacteria belonging to the genera Agrobacterium, Brevibacterium, Paracoccus, Brevundimonas, and Erythrobacter, green algae of the genus Haematococcus, and yeasts of the genus Phaffia. Carotenoids such as astaxanthin and adonixanthin are industrially produced by chemical synthesis, but due to increasing health and environmental awareness, natural products are in demand.

[0004] Among the above microorganisms, bacteria belonging to the genus Paracoccus have advantages such as high carotenoid productivity, fast growth rate, and ease of carotenoid extraction. An example of an astaxanthin-producing strain belonging to the genus Paracoccus is the E-396 strain (FERM BP-4283: dated April 27, 1993 (original deposit date), National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (6-1-1 Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Japan)) (Patent Document 2).

[0005] On the other hand, cis-isomerization of carotenoids is known to contribute to improving the extraction efficiency from bacterial cells, and it is also known that cis-isomers of carotenoids contribute to improving their bioavailability. However, when attempting to obtain cis-carotenoids from microorganisms, the carotenoids contained in the cells of microorganisms have the problem that the isomerization efficiency is low and the yield is low compared to isomerization of the carotenoid molecules themselves, due to incorporation into cellular tissues such as cell membranes and interactions with biopolymers such as lipids contained in the cells.

[0006] In addition, carotenoids generally have low solubility in water. While using water as an extraction solvent is preferable in terms of availability, ease of handling during production, environmental impact, and application to humans and animals, due to the low solubility mentioned above, it has been difficult to extract carotenoids with water-containing solvents, and the carotenoid yield has been low.

[0007] Patent Document 3 discloses a method for thermal isomerization of synthetic lycopene using methanol, which involves heat-treating lycopene molecules in a polar solvent to increase the amount of all-E-lycopene (trans isomer). However, in the method described in Patent Document 3, the starting material is not contained in a microorganism, and the effect of increasing the cis isomerization rate is not observed. Furthermore, the only extraction solvent shown to be effective in Patent Document 3 is methanol.

[0008] It has also been reported that an organic solvent in a supercritical or subcritical CO2 system is used to extract carotenoid pigments. Specifically, Patent Document 4 describes a method for extracting carotenoid pigments by contacting a plant-derived carotenoid pigment-containing material with an organic solvent in a supercritical or subcritical CO2 system. However, Patent Document 4 does not mention cis-isomerization.

[0009] Therefore, from the viewpoint of improving extraction efficiency from bacterial cells and improving absorption by the body, there remains a need for a technology for producing cis-carotenoids that can be applied even when they are contained in microorganisms. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-025712 [Patent Document 2] Japanese Patent Application Publication No. 08-009964 [Patent Document 3] Special Publication No. 2004-521931 [Patent Document 4] Japanese Patent Application Laid-Open No. 2007-046015 Summary of the Invention

[0011] The present inventors have now found that treating microbial cells containing trans-carotenoids in a subcritical fluid containing water improves the cis-isomerization ratio of carotenoids in a processed product of the microorganisms. The present invention is based on this finding.

[0012] Therefore, the present invention provides a method for increasing the cis-isomerization ratio of carotenoids in processed products of microorganisms.

[0013] The present invention includes the following inventions. [1] A method for isomerizing trans-carotenoids to cis-carotenoids, comprising treating a microorganism containing the trans-carotenoids in a subcritical fluid, the fluid containing water. [2] The method according to [1], wherein the temperature in the subcritical fluid treatment step is 160°C or higher. [3] The method according to [1] or [2], wherein the fluid further comprises ethanol. [4] The method according to [3], wherein the ethanol content is 20% by mass to 80% by mass relative to the total mass of the fluid. [5] The method according to any one of [1] to [4], wherein the fluid further contains at least one selected from an antioxidant and a vegetable oil. [6] The method according to any one of [1] to [5], wherein the antioxidant is at least one selected from the group consisting of ascorbic acid, α-tocopherol, dibutylhydroxytoluene, and propyl gallate, and the antioxidant is at least one selected from the group consisting of soybean oil and mustard oil. [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-carotenoid is at least one selected from the group consisting of trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin, and trans-β-cryptoxanthin. [9] A method for producing a processed product of microorganisms containing cis-carotenoids, comprising a step of treating microorganisms containing trans-carotenoids in a subcritical fluid, wherein the fluid contains water.

[10] The method described in [9], wherein the processed product of the microorganism comprises at least one selected from the group consisting of cis-astaxanthin, cis-adonirubin, and cis-adonixanthin.

[11] The method described in

[10] , wherein the cis-astaxanthin content relative to the total amount of astaxanthin in the processed product of the microorganism is 10 area% or more, the cis-adonirubin content relative to the total amount of adonirubin is 10 area% or more, or the cis-adonixanthin content relative to the total amount of adonixanthin is 10 area% or more.

[12] A processed product of a microorganism containing at least one cis-carotenoid selected from the group consisting of cis-astaxanthin, cis-adonirubin, and cis-adonixanthin, which satisfies at least one of the following (i) to (iii): (i) The content of endogenous cis-astaxanthin relative to the total amount of endogenous astaxanthin is 51% or more by area; (ii) the content of endogenous cis-adonirubin relative to the total amount of endogenous adonirubin is 57% or more by area; (iii) The content of endogenous cis-adonixanthin relative to the total amount of endogenous adonixanthin is 47% or more by area.

[13] The processed product according to

[12] , wherein all of the carotenoids in the processed product of the microorganism are endogenous.

[14] The processed product according to

[12] or

[13] , wherein the processed product of the microorganism is a dried product.

[15] The processed product according to any one of

[12] to

[14] , wherein the microorganism is Paracoccus carotinifaciens.

[0014] According to the present invention, trans-carotenoids can be efficiently isomerized to cis-carotenoids, and the cis-isomerization ratio of carotenoids in products processed by microorganisms can be improved. According to the present invention, the cis-isomerization ratio of carotenoids in products processed by microorganisms can be improved, and it is also possible to improve the yield of cis-carotenoids. Furthermore, according to the present invention, the cis-carotenoid content, cis-isomerization ratio and / or carotenoid residual rate of carotenoids in products processed by microorganisms can be improved, and it is also possible to improve the yield of cis-carotenoids. Furthermore, according to the present invention, it is advantageous in that the endogenous cis-carotenoid content can be improved in products processed by microorganisms containing cis-carotenoids. Specific Description of the Invention

[0015] The method of the present invention for isomerizing trans-carotenoids to cis-carotenoids comprises a step of treating a microorganism containing the trans-carotenoid in a subcritical fluid, and one of its characteristics is that the fluid contains water.

[0016] In this specification, when a numerical range is indicated using "to" it is intended to include both the lower and upper limits unless otherwise specified. For example, the expression "10 to 20" includes both the lower limit "10" and the upper limit "20". In other words, "10 to 20" has the same meaning as "10 or more and 20 or less."

[0017] Carotenoids According to one embodiment of the present invention, the carotenoid is not particularly limited as long as it does not interfere with the effects of the present invention, but examples include carotenes and xanthophylls, preferably xanthophylls. Specific examples of carotenoids in the present invention include xanthophylls such as astaxanthin, adonirubin, adonixanthin, zeaxanthin, β-cryptoxanthin, lutein, echinenone, canthaxanthin, 3-hydroxyechinenone, asteroidenone, violaxanthin, antheraxanthin, neoxanthin, fucoxanthin, peridinin, and rhodoxanthin, as well as carotenes such as lycopene and β-carotene, preferably astaxanthin, adonirubin, and adonixanthin. These carotenoids may be used alone or in combination of two or more. For example, a combination of astaxanthin, adonirubin, and adonixanthin may be used.

[0018] According to another embodiment of the present invention, the carotenoid of the present invention preferably further comprises at least one selected from the group consisting of canthaxanthin, asteroidenone, β-carotene, echinenone, and 3-hydroxyechinenone in addition to astaxanthin, adonirubin, and adonixanthin.More preferably, the carotenoid of the present invention further comprises canthaxanthin, asteroidenone, β-carotene, echinenone, and 3-hydroxyechinenone in addition to astaxanthin, adonirubin, and adonixanthin.

[0019] Carotenoids exist as isomers, with one or more conjugated double bonds in the center of the molecule being cis- or trans-isomers. Isomers in which one or more of the conjugated double bonds in the molecule are cis-form are called "cis-form" or "cis-type" carotenoids, while isomers in which all of the conjugated double bonds in the molecule are trans-form are called "trans-form," "trans-type," or "all-trans-type" carotenoids. When simply referring to "carotenoid," this term includes both cis- and trans-type carotenoids. The definition of cis and trans-isomers also applies to carotenoids such as astaxanthin, adonirubin, and adonixanthin.

[0020] Astaxanthin is a red pigment whose 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). Cis-astaxanthin includes 9-cis, 13-cis, 15-cis, di-cis, and 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]

[0021] The chemical formula of adonirubin 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: Furthermore, cis-adonirubin includes the 9-cis form, 13-cis form, 15-cis form, di-cis form, or combinations thereof. [ka]

[0022] The chemical formula of adonixanthin 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: Cis-adonixanthin includes the 9-cis form, 13-cis form, 15-cis form, di-cis form, or combinations thereof. [ka]

[0023] Furthermore, according to a preferred embodiment of the present invention, the carotenoid of the present invention includes one or more carotenoids selected from astaxanthin, adonirubin, and adonixanthin.

[0024] The carotenoid of the present invention may be in the form of a free carotenoid, a fatty acid ester, or a pharmaceutically acceptable salt, and is preferably in the free carotenoid form from the viewpoint of absorbability. The carotenoid may also be an optical isomer.

[0025] In the present invention, the carotenoid may be in the form of a pharmaceutically acceptable salt, and these salts are also included in the carotenoid of the present invention. In the present invention, the carotenoid may form a salt with an acid or a base. In the present invention, the pharmaceutically acceptable salt is not particularly limited as long as it forms a pharmaceutically acceptable salt with the carotenoid. Specific examples include hydrohalides (e.g., hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, bicarbonates, etc.), organic carboxylates (e.g., acetates, oxalates, maleates, tartrates, fumarates, citrates, etc.), organic sulfonates (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.), alkaline earth metal salts (e.g., magnesium salts, calcium salts, etc.), and the like, but are not limited to these.

[0026] The carotenoid of the present invention may also 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, and the 3S,3'S-isomer is preferred. The optical isomer of adonixanthin may be at least one selected from the group consisting of the 3S,3'R-isomer, the 3S,3'S-isomer, the 3R,3'S-isomer and the 3R,3'R-isomer, and is preferably the 3S,3'R-isomer.

[0027] According to one embodiment of the present invention, the carotenoid of the present invention (preferably, a trans-carotenoid) is preferably contained in a microorganism. Specifically, the trans-carotenoid of the present invention can be produced by a method using a microorganism (e.g., a fermentation method using a microorganism). Such microorganisms include bacteria, algae, and yeast. As the carotenoid-producing bacteria or yeast, bacteria or Phaffia yeast belonging to the genus Paracoccus, Sphingomonas, Brevundimonas, or Erythrobacter are preferably used, and more preferably bacteria belonging to the genus Paracoccus. Here, Paracoccus bacteria are preferably Paracoccus carotinifaciens, Paracoccus marcusii, Paracoccus haeundaensis, and Paracoccus zeaxanthinifaciens, with Paracoccus carotinifaciens being more preferred. Specific examples of Paracoccus strains include Paracoccus carotinifaciens strain E-396 and Paracoccus bacteria strain A581-1 (FERM BP-4671), and their mutants are also preferably used in the present invention.

[0028] Furthermore, the carotenoid-producing bacterium preferably uses a bacterium whose DNA nucleotide sequence corresponding to 16S ribosomal RNA has high homology (identity) with the nucleotide sequence of the E-396 strain. Here, "high homology" means that the DNA nucleotide sequence corresponding to 16S ribosomal RNA of the E-396 strain and the corresponding nucleotide sequence of the target bacterium are preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and most preferably 99% or more identical. Bacteria that are used have high homology with the nucleotide sequence of the E-396 strain. The nucleotide sequence of the DNA corresponding to 16S ribosomal RNA of the E-396 strain is listed, for example, in the sequence listing of WO 2010 / 044469. The nucleotide sequence of the DNA corresponding to 16S ribosomal RNA refers to a nucleotide sequence in which U (uracil) in the nucleotide sequence of 16S ribosomal RNA is replaced with T (thymine).

[0029] According to one embodiment of the present invention, mutant strains with improved carotenoid productivity can also be used as carotenoid-producing bacteria. Improved mutant strains can be known strains, such as strains with high astaxanthin productivity (e.g., strains described in JP 2001-95500 A). Furthermore, those skilled in the art can obtain mutant strains with improved carotenoid productivity through mutagenesis and screening using known techniques. The mutagenesis method is not particularly limited as long as it induces mutations. Examples of suitable methods include chemical methods using mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS), physical methods such as ultraviolet irradiation and X-ray irradiation, and biological methods using genetic recombination and transposons. While the microorganism to be mutagenized is not particularly limited, carotenoid-producing bacteria are preferred. Furthermore, mutant strains may be those resulting from naturally occurring mutations.

[0030] The method for culturing carotenoid-producing microorganisms (i.e., the method for producing carotenoids from carotenoid-producing microorganisms) and the method for processing the microorganisms are not particularly limited as long as they do not impair the effects of the present invention. For example, the methods for culturing carotenoid-producing bacteria and the methods for processing the bacteria are shown below.

[0031] Method for producing bacterial cells (culture) For example, carotenoid-producing bacteria are cultured according to the method of JP 2007-261972 A to prepare bacterial cells (culture).

[0032] In the present invention, the carotenoid-producing medium used for bacterial cultivation is not particularly limited as long as it allows carotenoid-producing bacteria to grow and produce carotenoids, but a medium containing a carbon source, a nitrogen source (specifically, an inorganic nitrogen source and / or an organic nitrogen source), inorganic salts, and, as necessary, vitamins, etc. is preferably used. It may also be preferable to further add amino acids, nucleic acid bases, etc. Other ingredients such as yeast extract, meat extract, and malt extract may also be added as appropriate.

[0033] 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. One or more of these carbon sources can be appropriately selected and used. Among these, glucose or sucrose is preferred. The amount of carbon source added to the medium before cultivation (initial medium) varies depending on the type of carbon source and can be adjusted as needed. Typically, the amount is 1 to 100 g, preferably 2 to 50 g, per 1 L of medium. In addition to adding the carbon source to the initial medium, it is also preferred to gradually or continuously add the carbon source during cultivation.

[0034] 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, and one or more of these can be appropriately selected and used. The amount added varies depending on the type of nitrogen source and can be adjusted appropriately, but is usually 0.1 g to 20 g, preferably 0.2 to 10 g, per 1 L of medium.

[0035] Examples of organic nitrogen sources include peptone, corn steep liquor (including filtered corn steep liquor), Pharmamedia, soybean meal, soybean flour, peanut meal, distiller's soluble, dry yeast, monosodium glutamate, etc., and one or more of these can be appropriately selected and used. The concentration of the added nitrogen source varies depending on the type of nitrogen source and can be adjusted appropriately, but is usually 0 to 80 g / L, preferably 0 to 40 g / L. Inorganic and organic nitrogen sources are usually added to the initial medium, but may also be added sequentially or continuously.

[0036] 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 ferrous 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 selenate; boric acid; and potassium iodide. One or more of these can be appropriately selected and used. The amount of inorganic salt added varies depending on the type of inorganic salt, and can be adjusted as needed; typically, it is 0.0001 to 15 g per 1 L of medium. Inorganic salts are usually added to the initial medium, but may also be added incrementally or continuously.

[0037] Examples of vitamins include cyanocobalamin, riboflavin, pantothenic acid, pyridoxine, thiamine, ascorbic acid, folic acid, niacin, p-aminobenzoic acid, biotin, inositol, and choline, and one or more of these can be appropriately selected and used. The addition rate varies depending on the type of vitamin and can be adjusted appropriately, but is usually 0.001 to 1000 mg per 1 L of medium, and preferably 0.01 to 100 mg. Vitamins are usually added to the initial medium, but may also be added sequentially or continuously.

[0038] The proportions of amino acids, nucleic acid bases, yeast extract, meat extract, malt extract, etc. added vary depending on the type of substance and can be adjusted appropriately, but are usually 0.2 g to 200 g, preferably 3 to 100 g, per 1 L of medium. The amino acids, etc. are usually added to the initial medium, but may also be added sequentially or continuously.

[0039] The carotenoid-producing medium used in the present invention is preferably sterilized before use in culturing bacteria. Sterilization can be carried out appropriately by those skilled in the art. For example, the medium in a suitable container can be heat-sterilized in an autoclave. Alternatively, the medium can be sterilized by filtration using a sterilizing filter.

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

[0041] Cultivation is carried out in an appropriate 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 to 80°C, preferably 20 to 35°C, and more preferably 25 to 32°C. Cultivation is carried out under aerobic conditions, usually for 1 to 20 days, preferably 2 to 12 days, and more preferably 3 to 9 days. Examples of aerobic conditions include shaking culture and aeration 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 agitation rotation speed, aeration volume, internal pressure, etc. The dissolved oxygen concentration is preferably controlled to 0.3 to 10 ppm, more preferably 0.5 to 7 ppm, and even more preferably 1 to 5 ppm.

[0042] According to one embodiment of the present invention, only the medium components can be removed from a culture such as a bacterial cell culture solution after the completion of the culture, based on known techniques. The bacterial cells may then be dried using a drum dryer. Drying methods that can be used include, in addition to the drum dryer, spray drying, granulation spray drying, freeze drying, and the like.

[0043] As described above, carotenoid-producing bacteria can be cultured and, in addition to a drum dryer, spray drying, granulation-type spray drying, freeze drying, etc. can be used.

[0044] According to another embodiment of the present invention, a concentrate containing carotenoids and bacterial cells can be separated from a culture obtained by culturing carotenoid-producing bacteria as described above by centrifugation, filtration, or decantation. The separation step can also be performed under acidic conditions. Here, in this specification, the term "culture" refers to any of the culture broth, culture supernatant, cultured bacterial cells, culture concentrate obtained by culturing, dried bacterial cells, and disrupted bacterial cells.

[0045] The culture can be subjected to the separation procedure as is, or it can be diluted with water before separation to enhance the removal of unnecessary components. Water can also be added during operations such as centrifugation, filtration, and decantation. After the culture is completed, heat sterilization can be performed to kill the cultured microorganisms before separation.

[0046] In the present invention, the method for separating bacterial cells can be a method based on sedimentation or particle size. Specifically, such a method may involve centrifugation, filtration, or decantation, either alone or in combination. Alternatively, the same type of separation may be repeated two or more times, such as by centrifuging once and then centrifuging the supernatant again to further recover carotenoids remaining in the supernatant. Centrifugation, filtration, or decantation can be appropriately performed by those skilled in the art using known techniques.

[0047] The culture concentrate obtained from the culture by the above separation method contains concentrated carotenoids and bacterial cells. The separation speed, separation strength, etc. can be suitably adjusted so that the culture concentrate has a viscosity and water content suitable for the next step. The culture concentrate can also be diluted by adding water, etc.

[0048] The microorganisms in the culture obtained as described above contain one or more carotenoids such as astaxanthin, adonirubin, and adonixanthin.

[0049] A step of treating a microorganism containing trans-carotenoids in a subcritical fluid According to a preferred embodiment of the present invention, the isomerization method of the present invention comprises a step of treating a microorganism containing trans-carotenoids in a subcritical fluid (hereinafter also referred to as subcritical fluid treatment or subcritical fluid treatment step), wherein the fluid contains water. Subcritical fluid treatment of a microorganism containing trans-carotenoids can be performed by contacting the microorganism with the subcritical fluid. Specifically, for example, when the fluid is water, the water in a microorganism-containing liquid (e.g., a culture concentrate) containing trans-carotenoids can be converted to a subcritical water state.

[0050] (fluid) The fluid is not particularly limited as long as it can be converted into a subcritical fluid state by heating and / or pressurization. The term "subcritical fluid" refers to a fluid that maintains a liquid state by pressurizing in a temperature range from the boiling point at atmospheric pressure to the critical temperature. The fluid preferably contains water from the viewpoints of availability, ease of handling during production, environmental impact, and application to humans and animals. Here, when the fluid is water, subcritical water refers to water that maintains a liquid state when pressurized in a range from the boiling point (100°C) at atmospheric pressure to the critical temperature (374.15°C). More preferably, the fluid further contains ethanol. The concentration of ethanol in the fluid is not particularly limited as long as it does not impair the effects of the present invention, but is, for example, 0.5 to 90% by mass, more preferably 10 to 85% by mass, even more preferably 20 to 80% by mass, and even more preferably 20 to 60% by mass. The inclusion of ethanol in the fluid is advantageous in that it improves the solubility of carotenoids and increases the cis-isomerization ratio. Furthermore, the fluid containing ethanol improves the solubility of carotenes, which is advantageous in that efficient isomerization can be achieved even at low temperatures.

[0051] (antioxidant) According to one embodiment of the present invention, the fluid may contain an antioxidant. The antioxidant of the present invention is not particularly limited, but is preferably one used as a food additive due to its ease of application to humans, animals, etc. The type of antioxidant can be appropriately selected depending on the type of carotenoid. Examples of antioxidants include ascorbic acid (VC), α-tocopherol, propyl gallate, butylhydroxyanisole (BHA), dibutylhydroxytoluene (BHT), ascorbyl palmitate (PAVC), coenzyme Q10, resveratrol, and curcumin, with ascorbic acid and α-tocopherol being preferred. These antioxidants may be used alone or in combination of two or more.

[0052] The concentration of the antioxidant in the fluid is not particularly limited as long as it does not interfere with the effects of the present invention, but is preferably 0.01 to 20 mass %, more preferably 0.1 to 10 mass %, and even more preferably 0.5 to 5 mass %.

[0053] (vegetable oil) According to one embodiment of the present invention, the fluid may contain a vegetable oil. The vegetable oil of the present invention is not particularly limited, and the type can be appropriately selected depending on the type of carotenoid. 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, and soybean oil and mustard oil are preferred. These vegetable oils may be used alone or in combination of two or more.

[0054] The concentration of the vegetable oil in the fluid is not particularly limited as long as it does not interfere with the effects of the present invention, but is preferably 0.01 to 20 mass %, more preferably 0.1 to 10 mass %, and even more preferably 0.5 to 5 mass %.

[0055] The heating temperature in the subcritical fluid treatment is not particularly limited as long as the effects of the present invention are achieved, but from the viewpoint of promoting the cis-isomerization reaction, it is, for example, 100°C or higher, preferably 120°C or higher, more preferably 140°C or higher, even more preferably 160°C or higher, and even more preferably 180°C or higher. From the viewpoint of suppressing decomposition of carotenoids, the heating temperature in the subcritical fluid treatment is, for example, 250°C or lower, preferably 240°C or lower, more preferably 220°C or lower, and even more preferably 200°C or lower.

[0056] The pressure in the subcritical fluid treatment is not particularly limited as long as the fluid is in a subcritical fluid state, but is preferably 1 MPa to 25 MPa, and more preferably 3 MPa to 18 MPa. A pressure equal to or higher than the lower limit of the above range can suitably convert the fluid into a subcritical fluid. Furthermore, a pressure equal to or lower than the upper limit of the above range can isomerize trans-carotenoids to cis-carotenoids at a lower temperature, which is preferable from the viewpoint of energy efficiency during production.

[0057] The time for the subcritical fluid treatment is not limited as long as the effects of the present invention are achieved, but may be, for example, 1 to 180 minutes, preferably 3 to 150 minutes, and more preferably 5 to 120 minutes. The treatment time may be appropriately set depending on the heating temperature. For example, when the heating temperature is 120 to 180°C, the treatment time is preferably 1 to 180 minutes. For example, when the heating temperature is higher than 180°C but not higher than 200°C, the treatment time is preferably 10 to 90 minutes. For example, when the heating temperature is higher than 200°C but not higher than 240°C, the treatment time is preferably 1 to 60 minutes.

[0058] According to a preferred embodiment of the present invention, the conditions for the subcritical fluid treatment are a heating temperature of 160 to 200° C., a pressure of 3 to 18 MPa, a time of 10 to 90 minutes, and the fluid containing ethanol together with water.

[0059] According to one embodiment of the present invention, the isomerization method of the present invention preferably includes a cooling step (also referred to as a cooling step) after the subcritical fluid treatment step. The cooling temperature in the cooling step may be lower than the lower limit of the heating temperature during the subcritical fluid treatment described above, and from the viewpoint of suppressing decomposition of carotenoids, may be, for example, 100°C or lower, preferably 60°C or lower, more preferably 40°C or lower, even more preferably 25°C or lower, and even more preferably 10°C or lower. The lower limit of the cooling temperature in the cooling step may be 0°C.

[0060] According to another embodiment of the present invention, the isomerization method of the present invention may further comprise, after the cooling step, a step of removing fluid from the microorganisms after the subcritical fluid treatment. The step of removing fluid may be, for example, a step of evaporating the fluid at 25 to 30°C under low pressure.

[0061] Microbial processed products containing cis-carotenoids According to another aspect of the present invention, a processed product of a microorganism containing cis-carotenoids is provided. Such a processed product of a microorganism contains at least cis-carotenoids. At least a portion of the cis-carotenoids are obtained by cis-isomerization of trans-carotenoids contained in the microorganisms. The processed product of a microorganism may contain any component other than cis-carotenoids and components derived from the microorganism. Examples of such any component include the above-mentioned antioxidants, vegetable oils, and residual components of fluids. Here, the processed product does not refer to the microorganism itself, but rather to a product in which the cis-carotenoid content has been increased by the above-mentioned isomerization method. Note that a processed product of a microorganism containing cis-carotenoids may also be referred to as a processed product of a microorganism containing cis-carotenoids, a cis-isomerized processed product, or a cis-isomerized product.

[0062] According to a preferred embodiment of the present invention, the cis-carotenoids contained in the microbial processed product containing cis-carotenoids include cis-carotenoids obtained by the above-mentioned isomerization method. Such cis-carotenoids are preferably cis-adonirubin, cis-adonixanthin, cis-astaxanthin, cis-zeaxanthin, and cis-β-cryptoxanthin, and more preferably cis-astaxanthin, cis-adonirubin, and cis-adonixanthin. These carotenoids may be used alone or in combination of two or more.

[0063] (cis-carotenoid content) In the microbial processed product containing cis-carotenoids of the present invention, the cis-carotenoid content (area %) (hereinafter simply referred to as cis-carotenoid content) when the total amount of carotenoids is taken as 100 area % is not particularly limited, but for example, it can be 10 area % or more, preferably 20 area % or more, more preferably 30 area % or more, even more preferably 40 area % or more, even more preferably 50 area % or more, and even more preferably 60 area % or more. The upper limit of the cis-carotenoid content is not particularly limited, but for example, it can be 90 area % or less, preferably 80 area % or less, and more preferably 70 area % or less. The cis-carotenoid content, the cis-isomerization ratio (described below), and the residual carotenoid rate in the processed microbial products of the present invention can be measured by HPLC (high-performance liquid chromatography) using a normal-phase column (preferably a silica column, more preferably a column with a particle size of 5 μm, a column length of 150 mm, and an internal diameter of 4.6 mm). Quantitation is based on the peak area of ​​each carotenoid isomer peak in the chromatogram. Such measurements can be easily performed using a commercially available HPLC device (e.g., manufactured by Shimadzu Corporation) and column (e.g., Luna, 5 μm, Silica (2), 100 Å (150 mm x φ4.6 mm) (manufactured by Phenomenex)). The above measurements can be performed under the following conditions. Apparatus: High-performance liquid chromatograph Prominence system (SPD-M20A, Shimadzu Corporation); Column: Two connected Luna, 5 μm, Silica (2), 100 Å (150 mm x φ4.6 mm) (Phenomenex); 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. HPLC samples can be prepared as follows. First, the processed microbial product is suspended in acetone and ultrasonicated at 10°C for 15 minutes, followed by filtration 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 resulting filtrate is evaporated under reduced pressure at 35°C to remove the solvent, after which it is dissolved in ethyl acetate / hexane (volume ratio 70:30) and filtered again through the filter to obtain an HPLC sample. The method for preparing HPLC samples from cultures is similar to the method for preparing HPLC samples from the processed microbial product described above.

[0064] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-astaxanthin, and the cis-astaxanthin content relative to the total amount of astaxanthin in the microbial processed product is, for example, 10 area% or more, preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 51 area% or more, and even more preferably 53 area% or more. The upper limit of the cis-astaxanthin content is not particularly limited, but is preferably 90 area% or less. The astaxanthin is preferably endogenous. Here, endogenous carotenoids and endogenous cis-carotenoids are those derived from endogenous carotenoids of microorganisms and include carotenoids (preferably cis-carotenoids) isomerized by the isomerization method of the present invention. The definitions of endogenous carotenoid and endogenous cis-carotenoid also apply to carotenoids such as astaxanthin, adonirubin, and adonixanthin.

[0065] According to one embodiment of the present invention, a processed product of a microorganism containing cis-carotenoids contains cis-adonirubin, and the cis-adonirubin content relative to the total amount of adonirubin in the processed product of the microorganism is, for example, 10 area% or more, preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 57 area% or more, and even more preferably 60 area% or more. There is no particular upper limit to the cis-adonirubin content, but it is preferably 90 area% or less. It is preferable that the adonirubin is endogenous.

[0066] According to one embodiment of the present invention, a processed product of a living organism containing cis-carotenoids contains cis-adonixanthin, and the cis-adonixanthin content relative to the total amount of adonixanthin in the processed product of the microorganism is, for example, 10 area% or more, preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 47 area% or more, and even more preferably 50 area% or more. The upper limit of the cis-adonixanthin content is not particularly limited, but is preferably 90 area% or less. The adonixanthin is preferably endogenous.

[0067] (cis isomerization ratio) The cis-isomerization ratio (hereinafter also referred to as cis-conversion ratio) of a microbially processed product containing cis-carotenoids of the present invention refers to the rate of increase in cis-carotenoids before and after isomerization (i.e., before the subcritical fluid treatment step and after the cooling step). When cis-isomerization is performed using a microorganism containing a carotenoid with a high concentration of trans-carotenoids (e.g., 93 area% or more, preferably 96 area% or more), the amount of cis-carotenoids before the isomerization is so small that it can be ignored. Therefore, the cis-isomerization ratio can be determined as the cis-carotenoid content of the resulting microbially processed product. That is, the cis-isomerization ratio (area %) is determined based on the peak area of ​​each carotenoid isomer peak in a chromatogram, similar to the cis-carotenoid content ratio. Specifically, when cis-isomerization is performed using a microorganism containing a carotenoid with a high concentration of trans-carotenoids, the cis-isomerization ratio can be determined by HPLC analysis of the microbially processed product using the following formula: Here, the peak area is the area value obtained by connecting the peak start point and end point when the peaks are separated, and is the area value obtained by vertically dividing the minimum value between the peaks when the peaks overlap.

number

[0068] The cis-isomerization ratio of carotenoids is not particularly limited, but may be, for example, 10 area% or more, preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 50 area% or more, and even more preferably 60 area% or more. The upper limit of the cis-carotenoid content is not particularly limited, but may be, for example, 90 area% or less, preferably 80 area% or less, and more preferably 70 area% or less.

[0069] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-astaxanthin, and the cis-isomerization ratio of astaxanthin in the microbial processed product (= (total peak areas of cis-astaxanthin / total peak areas of all astaxanthin) × 100) is, for example, 10 area% or more. The cis-isomerization ratio of astaxanthin is preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 51 area% or more, and even more preferably 53 area% or more. There is no particular upper limit to the cis-isomerization ratio of astaxanthin, but it is preferably 90 area% or less. The astaxanthin is preferably endogenous.

[0070] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-adonirubin, and the cis-isomerization ratio of adonirubin in the microbial processed product (= (total peak areas of cis-adonirubin / total peak areas of all adonirubins) × 100) is, for example, 10 area% or more. The cis-isomerization ratio of such adonirubin is preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 57 area% or more, and even more preferably 60 area% or more. There is no particular upper limit to the cis-isomerization ratio of adonirubin, but it is preferably 90 area% or less. It is preferable that the above-mentioned adonirubin is endogenous.

[0071] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-adonixanthin, and the cis-isomerization ratio of adonixanthin in the microbial processed product (= (total peak areas of cis-adonixanthin / total peak areas of all adonixanthin) × 100) is, for example, 10 area% or more. The cis-isomerization ratio of adonixanthin is preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 47 area% or more, and even more preferably 50 area% or more. There is no particular upper limit for the cis-isomerization ratio of adonixanthin, but it is preferably 90 area% or less. It is preferable that the adonixanthin is endogenous.

[0072] (Carotenoid residual rate) The residual carotenoid rate in the microbial processed product containing cis-carotenoids of the present invention means the ratio of the carotenoid content after the isomerization treatment (specifically, after the cooling step) to the carotenoid content before the isomerization treatment (specifically, before the subcritical fluid treatment step). The residual carotenoid rate can be calculated by HPLC analysis using the following formula:

number

[0073] The residual carotenoid content (hereinafter also referred to as residual rate) in the microbial processed product containing the cis-carotenoid of the present invention is not particularly limited, but may be, for example, 10 area% or more, preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 50 area% or more, and even more preferably 60 area% or more. The upper limit of the cis-carotenoid content is not particularly limited, but may be, for example, 100 area% or less, or 95 area% or less.

[0074] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-astaxanthin, and the residual astaxanthin rate in the microbial processed product (= (total peak areas of all astaxanthin after isomerization treatment / total peak areas of all astaxanthin before isomerization treatment) × 100) is, for example, 10 area% or more. The residual astaxanthin rate is preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 51 area% or more, and even more preferably 53 area% or more. The upper limit of the residual astaxanthin rate is not particularly limited, but is, for example, 100 area% or less. The astaxanthin is preferably endogenous.

[0075] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-adonirubin, and the residual adonirubin rate in the microbial processed product (= (total peak areas of all adonirubins after isomerization treatment / total peak areas of all adonirubins before isomerization treatment) x 100) is, for example, 10 area% or more. Such adonirubin residual rate is preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 57 area% or more, and even more preferably 60 area% or more. There is no particular limitation on the upper limit of the adonirubin residual rate, but it is, for example, 100 area% or less. It is preferable that the above adonirubin is endogenous.

[0076] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-adonixanthin, and the residual adonixanthin rate in the microbial processed product (= (total peak areas of all adonixanthin after isomerization treatment / total peak areas of all adonixanthin before isomerization treatment) × 100) is, for example, 10 area% or more. The residual adonixanthin rate is preferably 20 area% or more, more preferably 30 area% or more, even more preferably 40 area% or more, even more preferably 57 area% or more, and even more preferably 60 area% or more. The upper limit of the residual adonixanthin rate is not particularly limited, but is, for example, 100 area% or less. It is preferable that the adonixanthin is endogenous.

[0077] (Cis-carotenoid yield) The yield of cis-carotenoids (also referred to as cis-isomer yield) in the microbial processed product containing cis-carotenoids of the present invention is calculated by multiplying the cis-isomerization ratio by the residual carotenoid rate. The cis-carotenoid yield is not particularly limited, but may be, for example, 10% or more, preferably 20% or more, more preferably 30% or more, even more preferably 40% or more, and even more preferably 50% or more. The upper limit of the cis-carotenoid yield is not particularly limited, but may be, for example, 80% or less, or 70% or less.

[0078] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-astaxanthin, and the yield of cis-astaxanthin in the microbial processed product is calculated by multiplying the cis-isomerization ratio of the astaxanthin by the residual astaxanthin rate. The cis-astaxanthin yield is not particularly limited, but may be, for example, 5% or more, preferably 10% or more, more preferably 20% or more, even more preferably 30% or more, and even more preferably 40% or more. The upper limit of the cis-astaxanthin yield is not particularly limited, but may be, for example, 80% or less, or 70% or less.

[0079] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-adonirubin, and the yield of cis-adonirubin in the microbial processed product is calculated by multiplying the cis-isomerization ratio of the adonirubin by the residual adonirubin rate. The yield of cis-adonirubin is not particularly limited, but may be, for example, 5% or more, preferably 10% or more, more preferably 20% or more, even more preferably 30% or more, even more preferably 40% or more, and even more preferably 50% or more. The upper limit of the yield of cis-adonirubin is not particularly limited, but may be, for example, 80% or less, or 70% or less.

[0080] According to one embodiment of the present invention, a microbial processed product containing cis-carotenoids contains cis-adonixanthin, and the yield of cis-adonixanthin in the microbial processed product is calculated by multiplying the cis-isomerization ratio of the adonixanthin by the residual adonixanthin rate. The yield of cis-adonixanthin is not particularly limited, but may be, for example, 5% or more, preferably 10% or more, more preferably 20% or more, even more preferably 30% or more, and even more preferably 40% or more. The upper limit of the yield of cis-adonixanthin is not particularly limited, but may be, for example, 80% or less, or 70% or less.

[0081] According to a preferred embodiment of the present invention, all of the carotenoids in the microbial processed product containing cis-carotenoids are endogenous.

[0082] The processed product of the present invention, which is a microbial product containing cis-carotenoids, can be obtained by treating the microorganism containing trans-carotenoids in a subcritical fluid, followed by drying, pulverization, etc., as necessary. Therefore, examples of the processed product of the microorganism include a dried product and a pulverized product, and the dried product is preferred.

[0083] According to another aspect of the present invention, there is provided a composition containing cis-carotenoids derived from microorganisms. Such a composition containing cis-carotenoids is preferably a processed product of microorganisms containing cis-carotenoids.

[0084] According to one embodiment of the present invention, the microbially derived cis-carotenoid-containing composition of the present invention may contain optional components other than microbially derived components such as cis-carotenoids, in addition to the processed product of a microorganism containing cis-carotenoids. Examples of such optional components include the above-mentioned antioxidants, vegetable oils, and residual components of fluids. Furthermore, in addition to the optional components, the composition may optionally contain orally acceptable or pharmaceutically acceptable additives. Examples of such additives include solvents, solubilizers, dissolving agents, lubricants, emulsifiers, isotonicity agents, stabilizers, preservatives, antiseptics, surfactants, gelling agents, regulators, chelating agents, pH adjusters, buffers, excipients, thickeners, colorants, fragrances, sweeteners, and flavors.

[0085] According to a preferred embodiment of the present invention, the processed product or composition of the present invention may include a natural product, and more preferably, the processed product or composition of the present invention is solely a natural product. Here, a natural product is a product derived solely from a microorganism and does not contain additives such as preservatives (i.e., additive-free).

[0086] The processed microbial product or composition of the present invention can be used as, but is not limited to, feed (preferably, color-enhancing feed), food or beverage products (preferably, health foods), food additives, pharmaceuticals, quasi-drugs, or cosmetics, or as an ingredient of such feeds. The processed microbial product can be obtained by a cis-isomerization reaction in a subcritical fluid. As such a fluid, a fluid that is highly safe for animals such as humans (e.g., water) can be selected and is suitable for the above-mentioned applications.

[0087] According to one embodiment of the present invention, the processed product of a microorganism containing a cis-carotenoid or the composition containing a cis-carotenoid derived from a microorganism of the present invention can be produced by a production method comprising a step of treating a microorganism containing a trans-carotenoid in a subcritical fluid, wherein the fluid contains water.

[0088] The above-mentioned embodiment of the production method can be carried out in accordance with the description of the method of the present invention for isomerizing trans-carotenoids to cis-carotenoids or the processed product of a microorganism containing cis-carotenoids.

[0089] The method of ingestion or administration of the processed microbial product or composition of the present invention is not particularly limited, but includes injection such as drip infusion, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, oral, transmucosal, transdermal, intranasal, oral, intraperitoneal, etc., and preferably oral ingestion or administration.

[0090] The intake or administration amount of the processed microbial product or composition of the present invention is not particularly limited and can be determined depending on the formulation of the processed product or composition, the type and purity of the carotenoid, the type of subject, the age or weight of the subject, symptoms, the time of intake or administration, the form of the processed microbial product or composition, the method of intake or administration, and the combination of carotenoids or drugs other than the carotenoid of the present invention. The processed microbial product or composition of the present invention is preferably in the form of a daily intake unit so as to provide an effective amount for enhancing color, improving reproduction rate, enriching egg yolk nutrition, improving heat stress resistance, or enhancing immunity. For example, when the processed microbial product or composition of the present invention is orally ingested or administered, the processed microbial product can be prepared or incorporated into a composition so as to provide, for example, 0.01 to 1000 mg / kg body weight, preferably 0.05 to 500 mg / kg body weight, of carotenoid.

[0091] Furthermore, the daily intake or administration amount of the processed microbial product or composition of the present invention, like the intake or administration amount of the processed microbial product or composition described above, is appropriately selected depending on the formulation of the processed microbial product or composition. The daily intake or administration amount of the processed microbial product or composition of the present invention may be, for example, ingested or administered to a subject once or multiple times, but is preferably ingested or administered to a subject 1 to 5 times. Therefore, the daily intake or administration number of times of the composition of the present invention is 1 to 5 times per day, preferably 1 to 3 times per day, and more preferably once per day.

[0092] According to one embodiment of the present invention, the subject to which the processed microbial product or composition of the present invention is applied is not particularly limited as long as it does not interfere with the effects of the present invention, but is preferably fish, crustaceans, mammals, or birds, and more preferably salmon, trout, red sea bream, crab, shrimp, krill, ruminants such as cows and sheep, primates such as humans, and poultry such as dogs, cats, pigs, and chickens. The subject may be a healthy individual (healthy animal) or a patient (patient animal).

[0093] Another aspect of the present invention provides a method for enhancing the color of a subject, improving the reproductive rate of a subject, enhancing the nutritional value of egg yolks in a subject, improving the heat stress resistance of a subject, or improving the immune system of a subject, comprising administering or having a subject in need thereof ingest an effective amount of a processed product of a microorganism containing cis-carotenoids. Here, the term "effective amount" can be set to the same value as the carotenoid content in the processed product of the microorganism per daily intake unit. Here, "enhancing the color" includes adjusting the color of fish meat, egg yolk, etc. to suit consumer preferences.

[0094] Furthermore, according to another aspect of the present invention, there is provided use of a processed product of a microorganism containing a cis-carotenoid or a composition containing a cis-carotenoid derived from a microorganism for improving color, improving reproductive rate, enriching egg yolk nutritionally, improving heat stress resistance, or improving immunity.

[0095] Another aspect of the present invention provides the use of a processed microbial product containing cis-carotenoids as a composition for improving color, reproductive efficiency, nutritional value of egg yolk, heat stress resistance, or immunity.

[0096] Another aspect of the present invention provides the use of a processed microbial product containing cis-carotenoids in the manufacture of a composition for improving color, improving reproductive rate, enriching egg yolk, improving heat stress resistance, or improving immunity.

[0097] Another aspect of the present invention provides a processed product of a microorganism containing cis-carotenoids for improving color, reproductive rate, nutritional value of egg yolk, heat stress resistance, or immune function.

[0098] The above-mentioned methods, uses, and aspects of processed microbial products for color enhancement, etc. can be carried out in accordance with the description of the method of the present invention for isomerizing trans-carotenoids to cis-carotenoids or the description of processed microbial products containing cis-carotenoids. [Example]

[0099] The present invention will be described in more detail below with reference to Preparation Examples 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 in this specification are in accordance with JIS standards.

[0100] Preparation Example 1: Preparation of carotenoid-containing culture concentrate Paracoccus carotinifaciens E-396 strain (FERM BP-4283) was cultured according to the method described in Example 1 of Japanese Patent Application Laid-Open No. 2007-261972. Specifically, 10 ml of a 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. One loopful of E-396 strain (FERM BP-4283) was inoculated into the medium and cultured at 30°C for 6 days with reciprocal shaking at 300 rpm. The resulting culture solution was centrifuged and the culture supernatant was removed to obtain a carotenoid-containing culture concentrate (also referred to as a culture concentrate). The resulting carotenoid-containing culture concentrate was diluted appropriately with distilled water to obtain a sample for subcritical fluid treatment.

[0101] Test Example 1: Examination of temperature in the isomerization reaction (subcritical fluid treatment) of carotenoid-containing culture concentrate The subcritical fluid processing sample obtained in Preparation Example 1 was placed in a portable reactor (TPR3-VS2-120, Taiatsu Glass Industry Co., Ltd.), pressurized with nitrogen (4 MPa) and heated in an oil bath (140-240°C) to create subcritical water conditions, and an isomerization reaction (hereinafter also referred to as subcritical fluid processing) was carried out for 30 minutes. The heating temperature was varied between 140°C and 240°C. The reaction was then cooled in ice water to obtain a processed microbial product. The resulting processed microbial product was suspended in acetone, ultrasonicated at 10°C for 15 minutes, and then filtered through a PTFE filter (Osaka Chemical Co., Ltd., pore size 0.22 μm). The resulting 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 an HPLC sample (hereinafter also referred to as the sample). The obtained HPLC sample was analyzed by HPLC under the following analytical conditions, and based on the peak areas in the obtained chromatogram, the cis-isomerization ratio (also referred to as cis-conversion rate) of astaxanthin, adonirubin, and adonixanthin, the carotenoid residual rate (also referred to as residual rate), and the cis-isomer yield (also referred to as cis-yield) were calculated.

[0102] The HPLC was carried out under the following conditions. Apparatus: High-performance liquid chromatograph Prominence system (SPD-M20A, Shimadzu Corporation) Column: Luna, 5 μm, Silica (2), 100 Å (150 mm x φ4.6 mm) (Phenomenex), two columns connected together Mobile phase: hexane / ethyl acetate / acetone (70:20:10, v / v / v) Column temperature: 40℃ Flow rate: 1.2mL / min Detection wavelength: 470nm

[0103] The results are shown in Table 1. The values ​​for each sample are shown as the average and standard deviation (n=3). In the table, for astaxanthin, "all-E" indicates the trans form. "Other Z" indicates the cis form other than 9Z, 13Z, and 15Z. When the Paracoccus culture concentrate (carotenoid-containing culture concentrate) before subcritical fluid treatment was analyzed by HPLC under the above analytical conditions, the trans-isomer contents of astaxanthin, adonirubin, and adonixanthin were 93.2 area%, 96.7 area%, and 96.5 area%, respectively. [Table 1]

[0104] As is clear from Table 1, improvements in the cis-conversion rate, residual rate, and cis-isomer yield of carotenoids were observed at heating temperatures of 140°C or higher. Here, from the viewpoint of the cis-isomer yield of carotenoids, the heating temperature is preferably in the range of 160°C to 220°C, and more preferably in the range of 180°C to 200°C. Specifically, when the carotenoids are adonixanthin and astaxanthin, from the viewpoint of the cis-isomer yield, the heating temperature is preferably in the range of 160°C to 220°C, and more preferably in the range of 180°C to 200°C. When the carotenoid is adonirubin, from the viewpoint of the cis-isomer yield, the heating temperature is preferably in the range of 160°C to 220°C, and more preferably in the range of 200°C to 220°C.

[0105] Test Example 2: Investigation of pressure in subcritical fluid treatment of carotenoid-containing culture concentrate The same treatment as in Test Example 1 was carried out, except that the subcritical fluid treatment conditions were nitrogen pressures of 4 MPa, 8 MPa, 12 MPa, and 16 MPa, and the heating temperatures for each pressure were 180, 200, and 220°C, and the cis-conversion rate, residual rate, 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). [Table 2]

[0106] As is clear from Table 2, no significant changes were observed in the cis-conversion rate, residual rate, or cis-isomer yield of each carotenoid due to nitrogen pressure.

[0107] Test Example 3: Examination of the addition of ethanol in subcritical fluid treatment of carotenoid-containing culture concentrate The same treatment as in Test Example 1 was carried out, except that the subcritical fluid treatment conditions were a temperature of 160, 180, or 200°C and ethanol was added as a co-solvent to a final concentration of 0 to 50% by mass, and the cis-conversion rate, residual rate, and cis-isomer yield 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). [Table 3] JPEG0007805115000009.jpg253138JPEG0007805115000010.jpg254141

[0108] As is clear from Table 3, the addition of ethanol to subcritical water improved the cis-conversion rate for at least one carotenoid compared to subcritical fluid treatment using subcritical water alone. This is thought to be because the addition of ethanol improves the solubility of carotenoids, promoting cis-isomerization of carotenoids in the dissolved state. It is also thought that the improved solubility allows for efficient isomerization even at low temperatures. It is thought that subcritical fluid treatment at low temperatures suppresses thermal decomposition of carotenoids, resulting in a higher yield of cis-isomers.

[0109] Test Example 4: Examination of additives in subcritical fluid treatment of carotenoid-containing culture concentrate The same treatment as in Test Example 1 was performed, except that the subcritical fluid treatment conditions were that the additive was added to a final concentration of 1% by mass and the temperature was 200°C, and the cis-conversion rate, residual rate, and cis-isomer yield of astaxanthin, adonirubin, and adonixanthin were calculated. The additives used were ascorbic acid (Kanto Chemical Co., Ltd.), α-tocopherol (Tokyo Chemical Industry Co., Ltd.), dibutylhydroxytoluene (BHT) (Kanto Chemical Co., Ltd.), propyl gallate (Tokyo Chemical Industry Co., Ltd.), soybean oil (Kenkou Sarara, Ajinomoto Co., Inc.), and mustard oil (Tez, Recon Oil Industries Ltd.). The results are shown in Table 4. The values ​​for each sample are shown as the mean and standard deviation (n=3). [Table 4]

[0110] As is clear from Table 4, the addition of additives improved the cis-conversion rate and cis-isomer yield of carotenoids.

[0111] Test Example 5: Examination of heating time in subcritical fluid treatment of carotenoid-containing culture concentrate The same treatment as in Test Example 1 was performed, except that the subcritical fluid treatment conditions were varied as follows: heating time at a heating temperature of 180°C between 30 and 120 minutes, heating time at a heating temperature of 200°C between 15 and 60 minutes, or heating time at a heating temperature of 220°C between 5 and 45 minutes, and the cis-conversion rate, residual rate, and cis-isomer yield of astaxanthin, adonirubin, and adonixanthin were calculated. The results are shown in Table 5. The values ​​for each sample are shown as the mean and standard deviation (n=3). [Table 5]

[0112] As is clear from Table 5, an increase in the cis-conversion rate was observed by increasing the heating time. From the viewpoint of the cis-conversion rate of carotenoids, a heating time of 30 to 120 minutes is preferable at a heating temperature of 180°C, a heating time of 15 to 60 minutes is preferable at a heating temperature of 200°C, and a heating time of 5 to 45 minutes is preferable at a heating temperature of 220°C. Furthermore, as is clear from Table 5, the time required to achieve a high cis-isomer yield varies depending on the heating temperature. From the viewpoint of the carotenoid cis-isomer yield, it was considered that a range of 30 to 120 minutes is preferable at a heating temperature of 180°C, a range of 15 to 45 minutes is preferable at a heating temperature of 200°C, and a range of 5 to 30 minutes is preferable at a heating temperature of 220°C.

[0113] Test Example 6: Examination of the presence or absence of ethanol and additives in subcritical fluid treatment of carotenoid-containing culture concentrate (optimization study) The same treatment as in Test Example 1 was carried out, except that the subcritical fluid treatment conditions were a temperature of 160, 180, or 200°C and ethanol (final concentration: 50% by mass) and / or α-tocopherol (final concentration: 1% by mass), and the cis-conversion rate, residual rate, and cis-isomer yield of astaxanthin, adonirubin, and adonixanthin were calculated. The results are shown in Table 6. The values ​​for each sample are shown as the mean and standard deviation (n=3). [Table 6]

[0114] As is clear from Table 6, the addition of ethanol further improved the cis-conversion rate or cis-isomer yield of at least one carotenoid selected from astaxanthin, adonirubin, and adonixanthin at heating temperatures of 160°C, 180°C, and 200°C. In particular, at heating temperatures of 160°C and 180°C, a particular improvement was observed in the cis-conversion rate or cis-isomer yield of at least one carotenoid selected from astaxanthin, adonirubin, and adonixanthin.

Claims

1. A method for isomerizing trans-carotenoids to cis-carotenoids, comprising the step of treating a microorganism containing the trans-carotenoids in a subcritical fluid, The treatment is carried out at a heating temperature of 160 to 200°C, a pressure of 3 MPa to 18 MPa, and a time of 10 minutes to 90 minutes; the fluid comprises water; The trans-carotenoid is at least one selected from the group consisting of trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin, and trans-β-cryptoxanthin; The microorganism is at least one selected from bacteria belonging to the genus Paracoccus, Sphingomonas, Brevundimonas, or Erythrobacter, or Phaffia yeast. method.

2. The method of claim 1 , wherein the fluid further comprises ethanol.

3. The method according to claim 2, wherein the concentration of the ethanol is 20% by mass to 80% by mass relative to the total mass of the fluid.

4. The method according to any one of claims 1 to 3, wherein the fluid further comprises at least one selected from an antioxidant and a vegetable oil.

5. 5. The method of claim 4, wherein the antioxidant is at least one selected from the group consisting of ascorbic acid, α-tocopherol, dibutylhydroxytoluene, and propyl gallate, and the vegetable oil is at least one selected from the group consisting of soybean oil and mustard oil.

6. The method according to any one of claims 1 to 5, wherein the microorganism is Paracoccus carotinifaciens.

7. The method according to any one of claims 1 to 6, wherein the trans-carotenoid is at least one selected from the group consisting of trans-adonirubin, trans-adonixanthin, and trans-astaxanthin.

8. A method for producing a processed product of microorganisms containing cis-carotenoids, comprising a step of treating microorganisms containing trans-carotenoids in a subcritical fluid, The treatment is carried out at a heating temperature of 160 to 200°C, a pressure of 3 MPa to 18 MPa, and a time of 10 minutes to 90 minutes; the fluid comprises water; The trans-carotenoid is at least one selected from the group consisting of trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin, and trans-β-cryptoxanthin; The microorganism is at least one selected from bacteria belonging to the genus Paracoccus, Sphingomonas, Brevundimonas, or Erythrobacter, or Phaffia yeast. method.

9. The method according to claim 8, wherein the processed product of the microorganism comprises at least one selected from the group consisting of cis-astaxanthin, cis-adonirubin, and cis-adonixanthin.

10. The method according to claim 9, wherein the cis-astaxanthin content relative to the total amount of astaxanthin in the processed product of the microorganism is 10 area% or more, the cis-adonirubin content relative to the total amount of adonirubin is 10 area% or more, or the cis-adonixanthin content relative to the total amount of adonixanthin is 10 area% or more.

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