Method for isomerizing carotenoid

Abstract

This method for isomerizing a trans-carotenoid into a cis-carotenoid includes a step for performing microwave treatment on a mixture of: a raw material that contains a trans- carotenoid; and an additive that contains oils and fats and an emulsifier.

Description

Method for isomerizing carotenoids

[0001] The present invention relates to a method for isomerizing trans-carotenoids to cis-carotenoids.

[0002] Carotenoids are natural pigments widely found in nature, and examples of carotenoids include astaxanthin, adonirubin, adonixanthin, zeaxanthin, and β-cryptoxanthin. These carotenoids are known to have various physiological effects. Cis-carotenoids are more readily absorbed by the body than trans-carotenoids, and are expected to have superior effects than trans-carotenoids, making them suitable for use in foods, pharmaceuticals, and other applications.

[0003] Patent Document 1 describes a primary composition in which a specific cis isomer is enriched by subjecting a lycopene-containing carotenoid composition to heat treatment or microwave irradiation, and a method for producing the primary composition. Patent Document 2 describes a method for cis-isomerizing trans-carotenoids by heat treatment (particularly under subcritical conditions). Non-Patent Document 1 describes the stability (depletion and changes in isomer ratio) of krill-derived astaxanthin during processing steps including microwave drying.

[0004] However, the method described in Patent Document 1 is a method for cis-isomerizing lycopene using microwaves, and there are no examples of astaxanthin, so the effect of isomerization on trans-astaxanthin and other isomers is unclear. Furthermore, although the cis-isomer ratio achieved by this method is high at approximately 67%, it is easy to assume that decomposition also occurs simultaneously due to the high treatment intensity. Furthermore, the yield of cis-isomers is not shown, so its efficiency is unknown. Furthermore, the method described in Patent Document 2 is a method for isomerizing carotenoids under subcritical conditions, and the effect of microwave treatment is unclear.

[0005] In the method described in Non-Patent Document 1, krill (astaxanthin-containing plankton) is microwave-dried to obtain an astaxanthin-containing powder. However, as the microwave output is increased, the cis-astaxanthin obtained is approximately 35% at most, but the decomposition rate increases to approximately 80%, resulting in a low yield of cis-astaxanthin.

[0006] Furthermore, when attempting to obtain cis-carotenoids from microorganisms or algae, the carotenoids contained in the cells of the microorganisms or algae are incorporated into cellular tissues such as cell membranes, and due to interactions with biopolymers such as lipids contained in the cells, there are problems in that the isomerization efficiency is low and the yield is low compared to isomerizing carotenoid molecules.

[0007] JP 2012-211169 A JP 2023-16059 A

[0008] Effects of drying methods on the content, structural isomers, and composition of astaxanthin in Antarctic krill, ACS Omega 2019, 4, 19, 17972-17980

[0009] For these reasons, there has been a need to develop a method for stably and efficiently isomerizing carotenoids to the cis form.

[0010] As a result of intensive research conducted by the inventors to solve the above problems, they have completed the present invention by microwave treating a mixture of a raw material containing carotenoids and an additive containing an emulsifier and fats and oils.

[0011] That is, the present invention is as follows: [1] A method for isomerizing trans-carotenoids to cis-carotenoids, comprising a step of subjecting a mixture of a raw material containing trans-carotenoids and an additive containing fats and oils and an emulsifier to microwave treatment. [2] The method according to [1], wherein the content of the additive is 0.1% by mass or more and 50% by mass or less based on the total mass of the mixture. [3] The method according to [1], wherein the microwave output is 300 W or more. [4] The method according to [1], wherein the trans-carotenoid is at least one selected from trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin, and trans-β-cryptoxanthin. [5] The method according to [1], wherein the trans-carotenoid is derived from bacteria, yeast, algae, animals, or plants. [6] The method according to [5], wherein the bacterium belongs to the genus Paracoccus. [7] A method for producing cis-carotenoids, comprising the steps of: subjecting a mixture of a raw material containing trans-carotenoids and an additive containing fats and oils and an emulsifier to microwave treatment; and recovering the isomerized cis-carotenoids after the microwave treatment. [8] The method according to [7], wherein the amount of additive added is 0.1% by mass or more and 50% by mass or less based on the total mass of the mixture. [9] The method according to [7], wherein the microwave output is 300 W or more.

[10] The method according to [7], wherein the trans-carotenoid is at least one selected from trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin, and trans-β-cryptoxanthin.

[11] The method according to [7], wherein the trans-carotenoid is derived from bacteria, yeast, algae, animals, or plants.

[12] The method according to

[11] , wherein the bacterium belongs to the genus Paracoccus.

[0012] According to the present invention, trans-carotenoids can be isomerized to cis-carotenoids stably and in high yield.

[0013] FIG. 1 is a diagram showing the cis-form ratio, concentration, and residual rate of astaxanthin.

[0014] The scope of the present invention is not limited by these explanations, and appropriate modifications other than those exemplified below may be made without departing from the spirit of the present invention. The present invention relates to a method for isomerizing trans-carotenoids to cis-carotenoids, which comprises a step of subjecting a mixture of a raw material containing trans-carotenoids and an additive containing fats and oils and an emulsifier to microwave treatment. The present invention also relates to a method for producing cis-carotenoids, which comprises a step of recovering the isomerized cis-carotenoids.

[0015] 1. Overview By adding additives including fats and oils and emulsifiers to raw materials containing carotenoids and then subjecting them to microwave treatment, it is possible to achieve a high cis-isomer ratio while maintaining a high residual rate of carotenoids. Furthermore, because microwave treatment also doubles as a drying process, the isomerization and drying processes can be carried out simultaneously. This enables the efficient production of cis-isomer carotenoids, which was not possible with conventional technology.

[0016] Since cis-isomerization of carotenoids is believed to be promoted in a dissolved state, in the present invention, a high cis-isomer ratio was obtained by treating in the presence of fats and oils that have a high affinity for carotenoids. Furthermore, since the stability of carotenoids is improved in the presence of an emulsifier, a high residual rate was maintained by using an additive containing fats and oils and an emulsifier (e.g., powdered fats and oils), and carotenoids rich in cis-isomers were successfully obtained in high yield. Herein, a powdered composition containing fats and oils and an emulsifier is also referred to as "powdered fats and oils."

[0017] 2. Raw Materials Containing Trans Carotenoids In the present invention, examples of trans carotenoids to be converted to cis-form include trans astaxanthin, canthaxanthin, adonirubin, adonixanthin, β-carotene, β-cryptoxanthin, zeaxanthin, lutein, echinenone, 3-hydroxyechinenone, asteroidenone, β-apo-8'-carotenal, lactucaxanthin, tunaxanthin, capsanthin, capsorubin, violaxanthin, neoxanthin, fucoxanthin, siphonaxanthin, antheraxanthin, citranaxanthin, fucoxanthinol, amarousiaxanthin A, halocynthiaxanthin, diatoxanthin, peridinin, spirilloxanthin, rubixanthin, rhodoxanthin, and alloxanthin, and these may be used alone or in appropriate combination.

[0018] In the present invention, astaxanthin, adonirubin, adonixanthin, canthaxanthin, β-carotene, β-cryptoxanthin, zeaxanthin, lutein, echinenone, 3-hydroxyechinenone, asteroidenone, and the like are preferably used, and these can be used alone or in appropriate combination. Of the above carotenoids, the structure of astaxanthin is shown below.

[0019]

[0020] Of the compounds of the above structural formulas (A), (B), and (C), (A) is the trans-isomer of astaxanthin, and (B) and (C) are the cis-isomers of astaxanthin (B: (9Z)-astaxanthin, C: (13Z)-astaxanthin). The present invention has made it possible to efficiently isomerize trans-carotenoids to the cis-isomer under mild conditions, thereby successfully improving the yield of the cis-isomer.

[0021] In the present invention, the raw material containing trans-carotenoids is not particularly limited, and any raw material can be used. Examples include those derived from bacteria, yeast, algae, animals, or plants (see below). Furthermore, the trans-carotenoids may be chemically synthesized (e.g., commercially available products). Bacteria: bacteria belonging to the genus Paracoccus, Agrobacterium, Brevibacterium, Brevundimonas, or Erythrobacter, etc. Yeast: yeasts belonging to the genus Phaffia, Yarrowia, Rhodosporidium, Sporidiobolus, or Xanthophyllomyces, etc.

[0022] Algae: Haematococcus genus, Ulva genus, Chlorella genus, Dunaliella genus, Aurantiochytrium genus, Pinguiochrysis genus, Ochromonas genus, Phaeodactylum genus, Pleurochrysis genus, Chaetoceros Algae belonging to the genus Chaetoceros, Pavlova, Cladosiphon, Sargassum, Undaria, Chlamydomonas, Spirulina, Arthrospira, Codium, Euglena, or Symbiodinium, and the like.

[0023] Animals: shrimp, crab, salmon roe, krill, eggs, etc. Plants: green and yellow vegetables (pumpkin, carrot, angelica tree, komatsuna, shiso, spinach, corn, paprika, etc.), fruits (mango, papaya, persimmon, apricot, citrus fruits, wolfberry, rose hips, etc.), flowers (marigold, adonis, etc.), etc.

[0024] Examples of solvents that can be used when extracting carotenoids from the above-mentioned raw materials include lower alcohols, ketone solvents, hydrocarbon solvents, and ether solvents. As lower alcohols, ethanol, methanol, and isopropanol are preferably used, with ethanol being particularly preferred. As ketone solvents, acetone and ethyl acetate are preferably used. As hydrocarbon solvents, n-hexane and cyclohexane are preferably used. As ether solvents, for example, tetrahydrofuran (THF) is preferably used. Supercritical carbon dioxide and the like can also be used.

[0025] When a microorganism is used as a raw material in the present invention, wet bacterial cells obtained by subjecting the culture solution to a commonly known filtration method such as centrifugation can be used. The wet bacterial cells can also be dried by a commonly known drying method such as spray drying, fluidized bed drying, rotary drum drying, or freeze drying to obtain dried bacterial cells, which can be used as a raw material.

[0026] In the present invention, bacteria whose DNA base sequence corresponding to 16S ribosomal RNA is substantially homologous to the base sequence set forth in SEQ ID NO: 1 are preferably used, particularly in terms of growth rate and carotenoid productivity. "Substantially homologous" means that the sequence has, for example, 94% or more, 96% or more, or 98% or more homology, taking into account factors such as the frequency of errors in DNA sequencing. The E-396 strain (FERM BP-4283) is particularly preferred as such a bacterium. Alternatively, a high-carotenoid-producing strain selected by mutating the E-396 strain to improve carotenoid productivity may also be used. The E-396 strain has been internationally deposited with the International Patent Organism Depositary (IPOD) of the National Institute of Technology and Evaluation (NITE), Room 120, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture, 292-0818, Japan, as the international depositary authority, as follows: Identification: E-396, Accession number: FERM BP-4283, Original deposit date: April 27, 1993

[0027] 2. Additives Including Fats and Oils and Emulsifiers In the present invention, fats and oils are considered to be particularly effective in promoting isomerization, and emulsifiers are considered to be effective in improving stability. Therefore, in the present invention, fats and oils and emulsifiers can be used as additives.

[0028] Fats and oils are a type of lipid and basically refer to ester compounds of fatty acids and glycerin, but may also include fat-soluble nutrients. These ester compounds and fat-soluble nutrients are collectively referred to as "fats and oils" in this specification. The origin of fats and oils may be animal or vegetable oils. The number of carbon atoms in the fatty acids constituting the fats and oils used in the present invention is not particularly limited. For example, the fats and oils may be composed of short-chain fatty acids (carbon number less than 6), medium-chain fatty acids (carbon number 6-12), long-chain fatty acids (carbon number 13-21), or very-long-chain fatty acids (carbon number 22 or more), and the number of double bonds is also not limited. In the present invention, fatty acids with 8 to 24 carbon atoms are generally used. Furthermore, since carotenoids are reacted with fats and oils, edible fats and oil-soluble nutrients are preferred.

[0029] Fatty acids with 10 or more carbon atoms are also called higher fatty acids. Examples of higher fatty acids include lauric acid (which has 12 carbon atoms (written as "C12"; the same applies to other fatty acids)), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18), linoleic acid (C18), linolenic acid (C18), arachidonic acid (C20), eicosapentaenoic acid (C20), and docosahexaenoic acid (C22).

[0030] Examples of fats and oils that can be used in the present invention include olive oil, perilla oil, linseed oil, green nut oil, hemp seed oil, grape seed oil, soybean oil, corn oil, sesame oil, roasted sesame oil, mustard oil, rapeseed oil, argan oil, rice bran oil, peanut oil, safflower oil, macadamia oil, sunflower oil, palm oil, coconut oil, beef tallow, pork lard, etc. Examples of fat-soluble nutrients include fat-soluble vitamins such as vitamin A, vitamin D, vitamin E, and vitamin K.

[0031] Examples of emulsifiers include surfactants. Any of anionic surfactants, nonionic surfactants, zwitterionic surfactants, and cationic surfactants can be used as the surfactant. Examples of these surfactants are shown below.

[0032] <Anionic surfactants> Fatty acid surfactants (anionic): Sodium fatty acid, potassium fatty acid, sodium alpha sulfo fatty acid ester Linear alkyl benzene surfactants: Sodium linear alkyl benzene sulfonate Higher alcohol surfactants (anionic): Sodium alkyl sulfate, sodium alkyl ether sulfate Alpha olefin surfactants: Sodium alpha olefin sulfonate Normal paraffin surfactants: Sodium alkyl sulfonate

[0033] <Nonionic surfactants> Fatty acid surfactants (nonionic): sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters (polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), etc.), fatty acid alkanolamides, polyglycerin fatty acid esters, glycerin fatty acid esters, propylene glycol fatty acid esters Higher alcohol surfactants (nonionic): polyoxyethylene alkyl ethers Alkylphenol surfactants: polyoxyethylene alkylphenyl ethers Others: sodium caseinate, lecithin, lysolecithin, saponin, etc.

[0034] <Zwitterionic surfactants> Amino acid type: sodium alkylamino fatty acid Betaine type: alkyl betaine Amine oxide type: alkyl amine oxide

[0035] <Cationic (cationic) surfactants> Quaternary ammonium salts: alkyltrimethylammonium salts, dialkyldimethylammonium salts Although any of the above surfactants can be used, surfactants used for food are particularly preferred.

[0036] In the present invention, other additives may include excipients, antioxidants, flavorings, etc. Examples of excipients, antioxidants, and flavorings are shown below. Excipients: dextrin, maltodextrin, starch, trehalose, cellulose, lactose, etc. Antioxidants: vitamin E (tocopherol), vitamin C, rosemary extract, etc. Flavorings: butter flavor, lemon flavor, etc.

[0037] In the present invention, the mixing ratio of the additive is 0.1% by mass or more and 50% by mass or less with respect to the entire mixture of the raw material and the additive, for example, 1% by mass or more and 50% by mass or less with respect to the entire mixture, in another embodiment, 1% by mass or more and 30% by mass or less, and in yet another embodiment, 5% by mass or more and 30% by mass or less.

[0038] 3. Microwave Treatment There are no particular limitations on the device that generates microwaves, and a microwave heater / dryer (whether batch or continuous), a microwave laboratory machine, or the like may be used. Alternatively, a general household microwave oven may be used. Microwaves are electromagnetic waves with a frequency of 300 MHz to 300 GHz, and a frequency in the range of 900 MHz to 10 GHz (e.g., 2.45 GHz) is preferred.

[0039] The microwave energy has an output of at least 300 W, for example, an output of 300 W or more. In one embodiment of the present invention, the output is appropriately selected from the range of 300 to 1500 W. The microwave treatment time is 1 to 10 minutes, for example, 3 to 5 minutes. In this case, the applied energy is 18 to 900 kJ, for example, 90 to 300 kJ.

[0040] 4. Recovery of cis-carotenoids

[0041] In the present invention, a raw material containing trans-carotenoids is mixed with additives containing fats and oils and an emulsifier, and the mixture is subjected to microwave treatment to isomerize the trans-carotenoids to the cis-carotenoids, thereby obtaining carotenoids containing cis-carotenoids or a mixture of cis- and trans-carotenoids.

[0042] Microwave treatment promotes isomerization from trans to cis carotenoids. Microwave treatment simultaneously dries the carotenoids. Therefore, cis carotenoids can be recovered by simply recovering the dried product after microwave treatment. However, the dried product can also be further treated by conventional methods applied to carotenoids, such as chromatography, concentration, crystallization, filtration, and drying.

[0043] In the present invention, the cis-isomerization ratio (also referred to as the cis-form ratio) can be improved by at least 1.1 to 8.2 times compared to when no oils or fats and emulsifiers are added. For example, the improvement can be 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, or 1.6 times or more. The cis-isomerization ratio is calculated by the following formula.

[0044] By increasing the cis-isomerization ratio and the residual ratio, the cis-isomer can be obtained in high yield. The yield of the cis-isomer is calculated by the following formula: Yield = cis-isomerization ratio x residual ratio

[0045] The present invention will be described in more detail below with reference to the following examples. However, the scope of the present invention is not limited to these examples. [Example 1] Paracoccus sp. E-396 strain was cultured according to the method described in JP 2012-158569 A, and a concentrate containing carotenoids and bacterial cells was obtained by centrifugation. A concentrated culture solution of Paracoccus bacteria adjusted to an astaxanthin concentration of 2.5 g / L was mixed with powdered oil (5-30 wt%) and then subjected to microwave drying in a μReactor Ex (power: 500 W or 1000 W, time: 3 or 5 minutes). The dried powder after treatment was analyzed by HPLC, and the isomerization ratios and residual rates of astaxanthin, adonirubin, and adonixanthin were calculated. HPLC analysis was performed using a mixed solvent of hexane / ethyl acetate / acetone (70:20:10, v / v / v) as the mobile phase, two connected columns (Luna 5 μm Silica (2)), and a column temperature of 40°C.

[0046] <Conditions for Carotenoid Analysis by HPLC> Apparatus name: Shimadzu Corporation, Model No. LC-20AD, SPD-M20A Column used: Luna, 5 μm, Silica (2), 150 mm x 4.6 mm, two columns connected Mobile phase: n-hexane (Kanto Chemical) / ethyl acetate (Kanto Chemical) / acetone (Kanto Chemical) (70:20:10, v / v / v) Column temperature: 40°C Flow rate: 1.2 mL / min Detection wavelength: 470 nm

[0047] First, in this example, the cis-astaxanthin ratio, astaxanthin concentration, and residual astaxanthin rate were measured when fats and oils and emulsifiers were used alone. The results are shown in Figure 1. From Figure 1, it was considered that fats and oils are primarily effective in promoting isomerization, and emulsifiers are effective in improving stability. Next, the cis-astaxanthin ratio, concentration, and residual rate of astaxanthin, adonirubin, and adonixanthin were measured when a combination (mixture) of fats and oils and emulsifiers was used.

[0048] The results are shown in Tables 1 and 2.

[0049] The yield was calculated by multiplying the cis-carotenoid ratio (cis ratio) by the carotenoid residual ratio. In Tables 1 and 2, "Magic Fat 200" is a powdered oil made from palm oil, and "Emix AL" is an emulsified powder formulation made from tocopherol (a type of vitamin E). Furthermore, "Magical Ace N" is a powdered oil made from rapeseed oil. As shown in Tables 1 and 2, a higher cis-isomerization ratio was achieved when oils and fats were used compared to when they were not used. Furthermore, by using oils and fats in combination with an emulsifier, a high cis-isomerization ratio was achieved without the need for a carotenoid isolation process such as cell disruption.

[0050] SEQ ID NO: 1: n represents a, c, g or t (position: 1350).

Claims

1. A method for isomerizing trans-carotenoids to cis-carotenoids, comprising a step of subjecting a mixture of a raw material containing trans-carotenoids and additives including fats and oils and an emulsifier to microwave treatment.

2. The method according to claim 1, wherein the content of the additive is from 0.1% by mass to 50% by mass based on the total mixture.

3. The method according to claim 1, wherein the microwave output is 300 W or more.

4. The method according to claim 1, wherein the trans-carotenoid is at least one selected from trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin and trans-β-cryptoxanthin.

5. The method according to claim 1, wherein the trans-carotenoid is derived from bacteria, yeast, algae, animals or plants.

6. The method according to claim 5, wherein the bacterium belongs to the genus Paracoccus.

7. A method for producing cis-carotenoids, comprising the steps of: subjecting a mixture of a raw material containing trans-carotenoids and additives containing fats and oils and an emulsifier to microwave treatment; and recovering the isomerized cis-carotenoids after the microwave treatment step.

8. The method according to claim 7, wherein the amount of the additive added is 0.1% by mass or more and 50% by mass or less based on the total mixture.

9. The method according to claim 7, wherein the microwave output is 300 W or more.

10. The method according to claim 7, wherein the trans-carotenoid is at least one selected from trans-adonirubin, trans-adonixanthin, trans-astaxanthin, trans-zeaxanthin and trans-β-cryptoxanthin.

11. The method according to claim 7, wherein the trans-carotenoid is derived from bacteria, yeast, algae, animals or plants.

12. The method according to claim 11, wherein the bacterium belongs to the genus Paracoccus.

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