JP7674734B2 - Method for producing cis-carotenoid-containing composition - Google Patents

Description

The present invention relates to a method for producing a composition containing cis-carotenoids.

Carotenoids, which are widely present in nature, have strong antioxidant properties and a wide variety of colors, making them useful in a wide range of applications, including health foods, cosmetics, and food colorings. Generally, natural carotenoids exist as all-trans forms, in which all double bonds are trans. In recent years, it has been reported that some carotenoids (lycopene, astaxanthin) are more easily absorbed by the body in the cis form than in the trans form.

Patent Document 1 describes a method for cis-isomerizing lycopene, a type of carotenoid. In this method, the cis-isomerization of lycopene is promoted by heat treatment, microwave irradiation, and iodine-catalyzed photoisomerization treatment.

Patent Document 2 also describes a method for cis-isomerizing lycopene, a type of carotenoid. In this method, lycopene solution, in which lycopene and a specified photosensitizer are dissolved in a solvent consisting of ethyl acetate or hexane, is irradiated with light within a specified wavelength range to promote cis-isomerization of lycopene.

Special Publication No. 2007-522166 JP 2015-51930 A

However, because the conventional method is a batch-type process, the time required for cis-isomerization processing is long, and carotenoids could not be cis-isomerized in a short time. In addition, with batch-type processing, the decomposition of carotenoids progresses at the same time as the isomerization of carotenoids, resulting in a problem of a decrease in the residual rate of carotenoids after processing.

The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a method for producing a composition containing cis-form carotenoids that can efficiently cis-isomerize carotenoids and has a high residual rate of carotenoids after treatment.

It is known that natural carotenoids exist more abundantly as trans-carotenoids than as cis-carotenoids. The present inventors have separately found that cis-carotenoids are more easily absorbed and accumulated in animals than trans-carotenoids. Furthermore, it has been reported that cis-carotenoids have higher physiological activity (anti-inflammatory activity, anti-obesity activity, etc.) than trans-carotenoids. The present inventors have conducted extensive research to efficiently obtain useful cis-carotenoids, and have developed the production method of the present invention.

The method for producing a cis-carotenoid-containing composition of the present invention is a method for producing a cis-carotenoid-containing composition that continuously obtains a cis-carotenoid-containing composition, and includes a step of heating a mixture of a trans-carotenoid and an organic solvent flowing through a flow path, and a step of cooling the heated mixture flowing through the flow path after the heating step, in which the organic solvent is in a subcritical fluid state during the heating step.

This method for producing a cis-carotenoid-containing composition requires a short time for cis-isomerization treatment, and can sufficiently cis-isomerize carotenoids in a short time. In addition, this method for producing a cis-carotenoid-containing composition allows the mixture flowing through the flow path to be quickly heated and cooled. Therefore, compared to batch-type treatment, decomposition of carotenoids can be suppressed, and the residual rate of carotenoids after treatment can be improved.

Therefore, it is possible to provide a method for producing a composition containing cis-form carotenoids that can efficiently cis-isomerize carotenoids and has a high residual rate of carotenoids after treatment.

FIG. 1 is a schematic diagram showing a flow-type high-temperature high-pressure apparatus. 1 is a graph showing the effects of heating temperature and heating time in a method for producing a cis-lycopene-containing composition. 1 is a graph showing the effect of pressure in a method for producing a cis-lycopene-containing composition. 1 is a graph showing the effect of antioxidants in a method for producing a cis-lycopene-containing composition. 1 is a graph showing the effect of an isomerization-promoting catalyst in a method for producing a cis-lycopene-containing composition. 1 is a graph showing the effects of an antioxidant and an isomerization catalyst in a method for producing a cis-lycopene-containing composition. (a) is an HPLC chromatogram of lycopene contained in the raw material; (b) is an HPLC chromatogram of lycopene contained in the cis-lycopene-containing composition after treatment under optimal conditions. 1 is a graph showing the effect of heating temperature in a method for producing a cis-astaxanthin-containing composition. 1 is a graph showing the effect of pressure in a method for producing a cis-astaxanthin-containing composition. 1 is a graph showing the effects of heating temperature and heating time in a method for producing a cis-astaxanthin-containing composition. 1 is a graph showing the effect of an antioxidant in a method for producing a cis-astaxanthin-containing composition. 1 is a graph showing the effect of an isomerization-promoting catalyst in a method for producing a cis-astaxanthin-containing composition. 1 is a graph showing the effects of an antioxidant and an isomerization catalyst in a method for producing a cis-astaxanthin-containing composition. (a) is an HPLC chromatogram of astaxanthin contained in the raw material. (b) is an HPLC chromatogram of astaxanthin contained in the cis-astaxanthin-containing composition after treatment under optimal conditions.

A preferred embodiment of the present invention will now be described.
The heating time in the heating step is preferably 10 minutes or less, which is preferable in terms of increasing the residual rate of carotenoids.

The mixture may contain an antioxidant. This is preferable in terms of increasing the residual rate of carotenoids.

The mixture may contain an isomerization-promoting catalyst. This is preferable in terms of improving the cis-isomerization ratio.

The following describes embodiments of the present invention. In this specification, when a numerical range is described using "to", it is intended to include the lower limit and the upper limit unless otherwise specified. For example, the description "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".

1. Method for producing a cis-carotenoid-containing composition The method for producing a cis-carotenoid-containing composition is a method for continuously obtaining a cis-carotenoid-containing composition. The method for producing a cis-carotenoid-containing composition includes a step of heating a mixture of a trans-carotenoid and an organic solvent flowing through a flow path 12 (see FIG. 1 ), and a step of cooling the heated mixture flowing through the flow path 12 after the heating step. In the heating step, the organic solvent is in a subcritical fluid state.

(1) Raw Material The trans-carotenoid used as the raw material may be a trans-isomer of carotene or a trans-isomer of xanthophylls. The trans-carotenoid is preferably one or more trans-isomers selected from the group consisting of lycopene, β-carotene, astaxanthin, lutein, fucoxanthin, canthaxanthin, and zeaxanthin.

From the viewpoint of use in health foods, food dyes, and cosmetics, trans-carotenoid is more preferably trans-lycopene. Lycopene is a type of carotenoid represented by the chemical formula C ₄₀ H ₅₆ (molecular weight 536.87). Since lycopene has 11 conjugated double bonds, various cis isomers exist. In this application, an isomer in which at least one of the 11 conjugated double bonds of lycopene is a cis type is referred to as cis-lycopene, and an isomer in which all of the 11 conjugated double bonds are trans-type is referred to as trans-lycopene. When simply referring to "lycopene," both cis-lycopene and trans-lycopene are included. In this application, when the carbon skeleton is a cis type, its position is represented by a number. For example, in the HPLC chromatogram shown in FIG. 7(A), "all-trans" represents trans-lycopene. In the HPLC chromatogram shown in Fig. 7(B), "13cis" represents 13cis lycopene in which the carbon at position 13 is cis, and "9cis" represents 9cis lycopene in which the carbon at position 9 is cis.

In addition, it is more preferable that the trans-carotenoid is trans-astaxanthin from the viewpoint of use as a color enhancer for health foods, food dyes, cosmetics, and animal feed. Astaxanthin is a type of carotenoid represented by the chemical formula C ₄₀ H ₅₂ O ₄ (molecular weight 596.841). Astaxanthin is composed of a polyene chain consisting of nine conjugated double bonds and end groups (terminal groups) attached to both ends. In this application, an isomer in which at least one of the nine conjugated double bonds in the polyene chain is cis-type is referred to as cis-astaxanthin, and an isomer in which all are trans-type is referred to as trans-astaxanthin. In this application, when the carbon skeleton is cis-type, its position is represented by a number. For example, in the HPLC chromatogram shown in FIG. 14(A), "all-trans" represents trans-astaxanthin. In the HPLC chromatogram shown in Fig. 14(B), "9cis" represents 13cis astaxanthin in which the carbon at position 9 is cis. "13cis" represents 13cis astaxanthin in which the carbon at position 13 is cis.

Trans-carotenoids may be commercially available or may be produced by conventional chemical synthesis methods. In addition, trans-carotenoids may be chemically synthesized products, may be carotenoids derived from natural products, or may be a mixture of these. For food and beverage products, trans-carotenoids derived from natural products are more preferable from the viewpoint of safety. Raw materials containing trans-carotenoids may contain a certain proportion (e.g., 10% by mass or less) of cis-carotenoids.

The mixture is a mixture of trans-carotenoid and an organic solvent. The amount of trans-carotenoid can be, for example, 1 μg to 1000 μg per mL of organic solvent. The trans-carotenoid may be dissolved in the organic solvent, or may be dispersed in a state where it is partially or completely undissolved. The organic solvent is not particularly limited as long as it can be in a subcritical fluid state in the heating step. The term "subcritical fluid" refers to a fluid that is kept in a liquid state by pressurizing in a temperature range from the boiling point at normal pressure to the critical temperature. The organic solvent is preferably at least one selected from the group consisting of esters having 3 to 6 carbon atoms, alcohols having 1 to 4 carbon atoms, ketones having 3 to 9 carbon atoms, ethers having 2 to 8 carbon atoms, and alkanes having 5 to 8 carbon atoms. More specifically, the organic solvent is preferably at least one selected from the group consisting of ethyl acetate, methanol, ethanol, 2-propanol, acetone, diethyl ether, dimethyl ether, and hexane. Some commercially available trans-lycopene products are distributed in a form (extract) in which trans-lycopene is extracted from tomatoes or microorganisms with ethyl acetate. When the trans-carotenoid is trans-lycopene, the organic solvent is preferably ethyl acetate from the viewpoint of solubility. Also, some commercially available trans-astaxanthin products are distributed in a form (extract) in which trans-astaxanthin is extracted from microorganisms with ethanol or ethyl acetate. When the trans-carotenoid is trans-astaxanthin, the organic solvent is preferably ethanol or ethyl acetate from the viewpoint of solubility. As long as the mixture contains a trans-carotenoid and an organic solvent, it may further contain other components.

The mixture may contain an antioxidant. From the viewpoint of safety, the antioxidant is preferably one used as a food additive. The type of antioxidant can be appropriately selected according to the type of carotenoid. The antioxidant is more preferably one or more selected from the group consisting of α-tocopherol, butyl hydroxyanisole (BHA), dibutyl hydroxytoluene (BHT), ascorbyl palmitate (PAVC), and ascorbic acid (VC). When the trans-carotenoid is trans-lycopene, from the viewpoint of inhibiting the decomposition of lycopene, α-tocopherol, butyl hydroxyanisole (BHA), or dibutyl hydroxytoluene (BHT) are particularly preferable. When the trans-carotenoid is trans-astaxanthin, from the viewpoint of inhibiting the decomposition of astaxanthin, ascorbic acid (VC) is particularly preferable.
The concentration of the antioxidant is preferably 0.01 mM to 50 mM, more preferably 0.05 mM to 20 mM, and even more preferably 0.1 mM to 15 mM.

The mixture may contain an isomerization catalyst. From the viewpoint of safety, the isomerization catalyst is preferably a naturally derived component, particularly a food derived component. As the isomerization catalyst, isothiocyanates or polysulfides are preferable. More specifically, the isomerization catalyst is preferably one or more selected from the group consisting of allyl isothiocyanate, methyl isothiocyanate, ethyl isothiocyanate, sulforaphane, dimethyl disulfide, diallyl disulfide, and diallyl trisulfide.
The concentration of the isomerization-promoting catalyst is preferably 1 mM to 200 mM, more preferably 5 mM to 100 mM, and even more preferably 10 mM to 75 mM.

(2) Production method The production method of the cis-lycopene-containing composition is a method for continuously obtaining a cis-carotenoid-containing composition. Specifically, in this method, a mixture of trans-carotenoid and an organic solvent is continuously supplied, a cis-isomerization reaction of carotenoid occurs in the subcritical fluid sent in one direction, and the cis-carotenoid-containing composition is continuously recovered. That is, this method is a method in which supply, reaction, and recovery are performed simultaneously, and is also called a continuous method (flow method). This method can be performed, for example, using a flow-type high-temperature and high-pressure apparatus 10 shown in FIG. 1.

As shown in FIG. 1, the flow-type high-temperature high-pressure apparatus 10 includes a raw material supply section 11, a flow path 12, a high-pressure pump 13, a heating section 14, a cooling section 15, a back pressure valve 16, and a recovery section 17. The mixture supplied from the raw material supply section 11 flows through the flow path 12 and is sent to the recovery section 17. The flow path 12 is composed of a tube having high thermal conductivity and high rigidity, such as a metal. The inner diameter of the flow path 12 can be, for example, 0.1 mm to 120 mm. A predetermined pressure is applied to the mixture flowing through the flow path 12 in the section (pressure application section) from the high-pressure pump 13 to the back pressure valve 16. The flow-type high-temperature high-pressure apparatus 10 is provided with a heating section 14 and a cooling section 15 in the pressure application section. The heating section 14 heats the mixture from the outside of the flow path 12 by a heater or the like. The cooling section 15 cools the mixture from the outside of the flow path 12 by air cooling, water cooling, or the like. From the viewpoint of efficiently heating and cooling the mixture, the flow path 12 may be spiral or serpentine in the heating section 14 and the cooling section 15.

The heating step is a step of heating the mixture of trans-carotenoid and organic solvent flowing through the flow path 12. The heating step is performed while the mixture passes through the heating section 14. In the heating step, the mixture is heated in a temperature range from the boiling point to the critical temperature of the organic solvent. The boiling point of ethyl acetate is 77.10°C, and the critical temperature is 523.20K (250.15°C). The boiling point of ethanol is 78.32°C, and the critical temperature is 516.20K (243.05°C). In the heating step, a predetermined pressure is applied to the mixture. Then, when the temperature of the organic solvent reaches a predetermined temperature, the organic solvent becomes a subcritical fluid. The temperature and pressure at which the organic solvent becomes a subcritical fluid are determined by the type of organic solvent. Therefore, the heating temperature and pressure in the heating step are appropriately set according to the type of organic solvent.

The heating temperature in the heating step is preferably 40°C or higher, more preferably 60°C or higher, 80°C or higher, 100°C or higher, 120°C or higher, or 140°C or higher, from the viewpoint of promoting the cis-isomerization reaction. The heating temperature in the heating step is preferably 250°C or lower, more preferably 200°C or lower, 180°C or lower, or 160°C or lower, from the viewpoint of suppressing the decomposition of carotenoids. From these viewpoints, the preferred range of the heating temperature in the heating step can be set by appropriately combining the above lower and upper limits, and can be, for example, 40°C or higher and 250°C or lower. The above heating temperature is the temperature of the mixture. In this device 10, the set temperature of the heating unit 14 and the temperature of the mixture passing through the heating unit 14 are approximately the same, and the set temperature of the heating unit 14 can be the temperature of the mixture. The temperature of the mixture may also be predicted from the set temperature of the heater, for example, by investigating in advance the relationship between the set temperature of the heater and the temperature of the mixture in the flow path 12.

The present inventors have found that a sufficient cis-isomerization ratio can be achieved even with a short heating time by performing a cis-isomerization reaction in a subcritical fluid. The heating time in the heating step is preferably 5 seconds or more, and may be, for example, 10 seconds or more, 15 seconds or more, or 20 seconds or more. From the viewpoint of suppressing the decomposition of carotenoids, the heating time in the heating step is preferably 10 minutes or less, and more preferably 5 minutes or less, 3 minutes or less, 1 minute or less, or 50 seconds or less. From these viewpoints, the preferred range of the heating time in the heating step can be set by appropriately combining the above lower and upper limits, and can be, for example, 5 seconds or more and 10 minutes or less. This heating time can be controlled by appropriately adjusting the inner diameter of the flow path 12 and the flow rate of the mixture flowing through the flow path 12.

The pressure in the heating step is not particularly limited as long as the organic solvent is in a subcritical fluid state. The pressure in the heating step is higher than normal pressure, and is preferably 1 MPa to 100 MPa, more preferably 5 MPa to 50 MPa, and even more preferably 8 MPa to 15 MPa. If the pressure is equal to or higher than the lower limit of the above range, the organic solvent can be suitably made into a subcritical fluid. If the pressure is equal to or lower than the upper limit of the above range, a cis-carotenoid-containing composition can be obtained at a lower pressure, which is preferable from the viewpoint of energy efficiency during production.

The cooling step is a step of cooling the heated mixture flowing through the flow path 12 after the heating step. The cooling step is carried out while the mixture passes through the cooling section 15. The cooling temperature in the cooling step may be lower than the lower limit of the heating temperature described above, and is preferably 100°C or lower, more preferably 60°C or lower, and even more preferably 40°C or lower, from the viewpoint of suppressing decomposition of carotenoids. The lower limit of the cooling temperature in the cooling step is, for example, -10°C. The cooling temperature can be controlled, for example, by appropriately adjusting the temperature of the water or ethylene glycol aqueous solution used for cooling.

The method for producing a cis-carotenoid-containing composition may further include a step of removing the organic solvent from the mixture after the cooling step. The step of removing the organic solvent may be, for example, a step of evaporating the organic solvent at room temperature (25°C) under low pressure.

2. Cis-carotenoid-containing composition The cis-carotenoid-containing composition contains at least cis-carotenoid. At least a part of the cis-carotenoid is obtained by cis-isomerization of trans-carotenoid contained in the raw material. The cis-carotenoid-containing composition may contain any component other than the cis-carotenoid. Examples of such any component include the above-mentioned antioxidants, isomerization-promoting catalysts, residual components of organic solvents, etc.

In the cis-carotenoid-containing composition, the cis-carotenoid content (mass%) when the total carotenoid is taken as 100% by mass is not particularly limited. Hereinafter, the cis-carotenoid content when the total carotenoid is taken as 100% by mass is also simply referred to as the cis-carotenoid content. The cis-carotenoid content is preferably 10% by mass or more, more preferably 20% by mass or more, more preferably 30% by mass or more, more preferably 40% by mass or more, more preferably 50% by mass or more, and even more preferably 60% by mass or more. The upper limit of the cis-carotenoid content is not particularly limited, but is usually 90% by mass or less, and may be 85% by mass or less, 80% by mass or less, or 70% by mass or less. When the cis-carotenoid contained in the cis-carotenoid-containing composition is cis-lycopene, the cis-carotenoid content may be 70% by mass or more, or even 75% by mass or more.
In the present application, the carotenoid content and the cis-carotenoid content can be measured by HPLC (high performance liquid chromatography) using a reverse phase column or a normal phase column. The amount is determined based on the peak area of each carotenoid isomer peak in a chromatogram.

The cis-isomerization ratio means the increase rate of cis-carotenoid before and after the isomerization treatment (before the heating step and after the cooling step). When the cis-isomerization treatment is performed using a carotenoid with a high concentration (e.g., 97% by mass or more) of trans-carotenoid content, the amount of cis-carotenoid before the isomerization treatment is small and negligible. Therefore, the cis-isomerization ratio can be determined as the cis-carotenoid content of the obtained cis-carotenoid-containing composition. Specifically, when the cis-isomerization treatment is performed using a carotenoid with a high concentration of trans-carotenoid content, the cis-isomerization ratio can be determined by the following formula by HPLC analysis of the cis-carotenoid-containing composition.

The cis-isomerization ratio is preferably 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more. The upper limit of the cis-isomerization ratio is not particularly limited, but is usually 90% or less, and may be 85% or less, 80% or less, or 70% or less. When trans-lycopene is cis-isomerized, the cis-isomerization ratio may be 70% or more, or even 75% or more. If the cis-isomerization ratio is equal to or greater than the above lower limit, it is preferable from the viewpoint of ensuring the cis-carotenoid content in the carotenoid in the cis-carotenoid-containing composition. If the cis-isomerization ratio is equal to or greater than the above lower limit, it is preferable from the viewpoint of ensuring the cis-carotenoid content in the cis-carotenoid-containing composition.

The residual carotenoid ratio means the ratio of the carotenoid content after the isomerization treatment (after the cooling step) to the carotenoid content before the isomerization treatment (before the heating step). The residual carotenoid ratio can be calculated by HPLC analysis using the following formula.

The carotenoid residual rate is preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more. The upper limit of this carotenoid residual rate is 100%, and it may be 95% or less. If the carotenoid residual rate is equal to or greater than the above lower limit, this is preferable from the viewpoint of ensuring the carotenoid content of the cis-carotenoid-containing composition and further the cis-carotenoid content in the carotenoid.

The cis-carotenoid-containing composition is a material containing cis-carotenoids that are highly absorbable and highly accumulating in the body of animals, and is suitable for use in foods and beverages, cosmetics, pharmaceuticals, and animal feed. The cis-carotenoid-containing composition undergoes a cis-isomerization reaction in a subcritical fluid, making it possible to select an organic solvent that is highly safe for humans, making it suitable for the above uses.

3. Effects of the Present Embodiment As described above, according to the present embodiment, the time required for the cis-isomerization treatment is short, and carotenoids can be sufficiently cis-isomerized in a short time. Furthermore, according to the present embodiment, the mixture flowing through the flow path 12 can be quickly heated and cooled. Therefore, compared to the case of batch-type treatment, decomposition of carotenoids can be suppressed, and the residual rate of carotenoids after treatment can be improved. Therefore, according to the present embodiment, carotenoids can be efficiently cis-isomerized, and a method for producing a cis-carotenoid-containing composition having a high residual rate of carotenoids after treatment can be provided.

This method, based on flow chemistry, is also a useful tool from the perspective of green and sustainable chemistry.

In addition, according to this embodiment, a high cis-isomerization ratio can be achieved. Therefore, the yield of cis-carotenoids, calculated by multiplying the amount of trans-carotenoids in the raw material by the cis-isomerization ratio and the carotenoid residual rate, can be sufficiently ensured.

The following provides a more detailed explanation using examples.

1. Production of cis-lycopene-containing composition As trans-lycopene, lycopene purified from tomato oleoresin (Lycored Co., Ltd., Lyc-O-Mato (registered trademark) 15%) was used. The trans-lycopene content of this lycopene was 97.0 mass% or more. FIG. 7(A) shows a chromatogram of this lycopene (raw material). Ethyl acetate was used as the organic solvent. Lycopene was dissolved in ethyl acetate to obtain a mixed solution with a total lycopene concentration of 5.0 μg/ml. A tube (SUS-316, Shimadzu Corporation) with an inner diameter of 0.5 mm was used for the flow path. The heating temperature in the heating step was 80° C. to 180° C. The pressure in the heating step was 10 MPa to 40 MPa. Under these heating temperature and pressure conditions, ethyl acetate exists as a subcritical fluid. Further, α-tocopherol (Tokyo Chemical Industry Co., Ltd.), butyl hydroxyanisole (BHA, Tokyo Chemical Industry Co., Ltd.), dibutyl hydroxytoluene (BHT, Kanto Chemical Co., Ltd.), and ascorbyl palmitate (PAVC, Fujifilm Wako Pure Chemical Industries Co., Ltd.) were used as antioxidants. The final concentration of each antioxidant was adjusted to 0 mM to 10 mM. Allyl isothiocyanate (AITC, Kanto Chemical Co., Ltd.) was used as an isomerization catalyst. The final concentration of the isomerization catalyst was adjusted to 0 mM to 100 mM. The mixture after the heating step and the cooling step was evaporated to dryness under reduced pressure at 30°C to obtain a cis-lycopene-containing composition. The obtained cis-lycopene-containing composition was dissolved in hexane to obtain a sample for HPLC. The obtained sample was subjected to normal phase HPLC analysis under the conditions described below.

2. Production of cis-astaxanthin-containing composition Astaxanthin manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used as trans-astaxanthin. The trans-astaxanthin content of this astaxanthin was 97.0% by mass or more. FIG. 14 (A) shows a chromatogram of this astaxanthin (raw material). Ethanol was used as the organic solvent. Astaxanthin was dissolved in ethanol so that the total astaxanthin concentration was 2.5 μg/ml to obtain a mixed solution. A tube (SUS-316, manufactured by Shimadzu Corporation) with an inner diameter of 0.5 mm was used for the flow path. The heating temperature in the heating step was 60° C. to 200° C. The pressure in the heating step was 10 MPa to 40 MPa. Under these heating temperature and pressure conditions, ethanol exists as a subcritical fluid. Furthermore, ascorbic acid (VC, manufactured by Kanto Chemical Co., Ltd.), α-tocopherol (α-Tocopherol, manufactured by Tokyo Chemical Industry Co., Ltd.), and dibutylhydroxytoluene (BHT, manufactured by Kanto Chemical Co., Ltd.) were used as antioxidants. Each antioxidant was mixed to a final concentration of 0 mM to 20 mM. Allyl isothiocyanate (AITC, manufactured by Kanto Chemical Co., Ltd.) was used as an isomerization catalyst. The isomerization catalyst was mixed to a final concentration of 0 mM to 100 mM. The mixture after the heating step and the cooling step was evaporated to dryness under reduced pressure at 30°C to obtain a cis-astaxanthin-containing composition. The obtained cis-astaxanthin-containing composition was dissolved in ethyl acetate/hexane (volume ratio 70:30) to obtain a sample for HPLC. The obtained sample was subjected to normal phase HPLC analysis under the conditions described below.

3. Production of cis-β-carotene-containing composition As trans-β-carotene, β-carotene manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used. The trans-β-carotene content of this β-carotene was 97.9% by mass or more. Ethyl acetate was used as the organic solvent. β-carotene was dissolved in ethyl acetate to obtain a mixed solution with a total β-carotene concentration of 100 μg/ml. A tube (SUS-316, manufactured by Shimadzu Corporation) with an inner diameter of 0.5 mm was used for the flow path. The heating temperature in the heating step was 140° C. to 200° C. The pressure in the heating step was 10 MPa. Under the heating temperature and pressure conditions, ethyl acetate exists as a subcritical fluid. Furthermore, α-tocopherol (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the antioxidant. The antioxidant was mixed to a final concentration of 1 mg/mL. The mixture after the heating and cooling steps was evaporated to dryness under reduced pressure at 30° C. to obtain a cis-β-carotene-containing composition. The obtained cis-β-carotene-containing composition was dissolved in methanol/tert-butyl methyl ether/water (volume ratio 60:35:5) to obtain a sample for HPLC. The obtained sample was subjected to reversed-phase HPLC analysis under the conditions described below.

4. Conditions for HPLC Analysis (1) Conditions for normal phase HPLC analysis of cis-lycopene-containing composition Apparatus: High performance liquid chromatograph Prominence system (SPD-M20A, manufactured by Shimadzu Corporation)
Column: Nucleosil 300-5 (length: 250 mm x 3, inner diameter: 4.6 mm, particle size: 5 μm, manufactured by GL Sciences Inc.)
Mobile phase: hexane (containing 0.075% DIPEA (diisopropylethylamine))
Flow rate: 1.0mL/min
Detection wavelength: 460 nm
Column temperature: 40°C

(2) Conditions for normal phase HPLC analysis of cis-astaxanthin-containing composition Apparatus: High performance liquid chromatograph Prominence system (SPD-M20A, manufactured by Shimadzu Corporation)
Column: Penomenex silica gel luna (2) (length: 150 mm x 2, inner diameter: 4.6 mm, particle size: 5 μm, manufactured by Penomenex)
Mobile phase: hexane/ethyl acetate/acetone (volume ratio 70:20:10)
Flow rate: 1.2mL/min
Detection wavelength: 470 nm
Column temperature: 40°C

(3) Conditions for reversed-phase HPLC analysis of cis-β-carotene-containing composition: Apparatus: High-performance liquid chromatograph Prominence system (SPD-M20A, manufactured by Shimadzu Corporation)
Column: YMC Carotenoid (length: 250 mm x 3, inner diameter: 4.6 mm, particle size: 5 μm, manufactured by YMC Co., Ltd.)
Mobile phase: methanol/tert-butyl methyl ether/water (volume ratio 60:35:5)
Flow rate: 1.0mL/min
Detection wavelength: 450 nm
Column temperature: 40°C

The cis-isomerization ratio (%) and carotenoid residual ratio (%) of each sample were calculated based on the peak area in the chromatogram obtained by HPLC analysis. Measurements of the cis-isomerization ratio and carotenoid residual ratio were performed three times for each sample. Each measurement was analyzed using Tukey's test with a significance level of 5%.

5. Results and Discussion The measurement results of the cis-isomerization ratio (%) and the carotenoid remaining ratio (%) are shown in Tables 1 to 16, Figures 2 to 6, and 8 to 13. Tables 1 to 6 and Figures 2 to 6 show the measurement results for the cis-lycopene-containing composition, and Tables 7 to 12 and Figures 8 to 13 show the measurement results for the cis-astaxanthin-containing composition. The measured values in the tables and graphs are the average values of three measurements. In the tables, "-" indicates that no measurement was performed, and "ND" indicates that no peak indicating carotenoid was detected. In the tables and figures, "cis ratio (%)" and "Total Z-isomer Ratio (%)" represent the cis-isomerization ratio (%), and "remaining ratio (%)" and "Remaining ratio (%)" represent the carotenoid remaining ratio (%).

(1) Effect of heating temperature and heating time on cis-lycopene-containing composition Tables 1, 2, and Fig. 2 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at heating times of 0.25 minutes (15 seconds) to 4 minutes, heating temperatures of 80°C to 180°C, and pressures of 10 MPa. The higher the heating temperature, the more rapidly cis-isomerization was promoted, but the decomposition of lycopene was also promoted. It was suggested that the shorter the heating time, the more the decomposition of lycopene could be suppressed.

(2) Effect of pressure on cis-lycopene-containing composition Table 3 and Figure 3 show the measurement results of the cis-isomerization ratio and carotenoid residual ratio at heating temperatures of 80°C to 160°C, heating times of 1 minute, and pressures of 10 MPa to 40 MPa. At heating temperatures of 80°C and 120°C, the above pressures did not affect the cis-isomerization ratio. At a heating temperature of 160°C, the higher the pressure, the more accelerated the decomposition of lycopene.

(3) Effect of antioxidants on cis-lycopene-containing composition Table 4 and Figure 4 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at antioxidant concentrations of 0 mM to 10 mM, heating temperature of 160°C, heating time of 1 minute, and pressure of 10 MPa. α-Tocopherol, BHA, and BHT inhibited the decomposition of lycopene accompanying the isomerization treatment. PAVC was not confirmed to have an effect of inhibiting the decomposition of lycopene under the above conditions.

(4) Effect of isomerization catalyst on cis-lycopene-containing composition Table 5 and Figure 5 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at isomerization catalyst (catalyst) 0 mM to 100 mM, heating temperatures of 80°C and 120°C, heating time of 1 minute, and pressure of 10 MPa. The addition of the isomerization catalyst, especially at 50 mM or more, promoted the cis-isomerization reaction. The addition of a low concentration of about 1 mM to 10 mM was poor in promoting the cis-isomerization reaction. At 25 mM or more, the decomposition of lycopene was also promoted as the amount of isomerization catalyst added increased.

(5) Effects of antioxidant and isomerization catalyst on cis-lycopene-containing composition Table 6 and FIG. 6 show the measurement results of the cis-isomerization ratio and carotenoid residual ratio at 0 mM to 1 mM of antioxidant (α-tocopherol, αTC), 0 mM to 50 mM of isomerization catalyst (catalyst), heating temperature of 160°C, heating time of 15 seconds and 30 seconds, and pressure of 10 MPa. It was confirmed that lycopene can be cis-isomerized efficiently in a short time by using antioxidant and catalyst in combination. At a heating temperature of 160°C, heating time of 30 seconds, catalyst 50 mM, and antioxidant 1 mM, a cis-isomerization ratio of 80.7% and a carotenoid residual ratio of 92.0% were achieved. FIG. 7(B) shows a chromatogram of the cis-lycopene-containing composition obtained under these conditions.

(6) Effect of heating temperature on cis-astaxanthin-containing composition Table 7 and Fig. 8 show the measurement results of the cis-isomerization rate and the carotenoid residual rate at a heating time of 5 minutes, a heating temperature of 60°C to 180°C, and a pressure of 10 MPa. From these results, it was found that the higher the heating temperature is between 60°C and 160°C, the more the cis-isomerization is promoted, but the decomposition of astaxanthin is also promoted.

(7) Effect of Pressure on Cis-Astaxanthin-Containing Composition Table 8 and Figure 9 show the measurement results of the cis-isomerization ratio and carotenoid residual ratio at heating temperatures of 80°C to 160°C, heating times of 5 minutes, and pressures of 10 MPa to 40 MPa. The above pressures did not affect the cis-isomerization ratio and carotenoid residual ratio (decomposition). Note that, for lycopene, the higher the pressure at 160°C, the more accelerated the decomposition, but for astaxanthin, the decomposition was not accelerated even at 160°C.

(8) Effect of heating temperature and heating time on cis-astaxanthin-containing composition Tables 9, 10, and Fig. 10 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at heating times of 0.25 minutes (15 seconds) to 5 minutes, heating temperatures of 140°C to 200°C, and pressures of 10 MPa. The higher the heating temperature, the more cis-isomerization was promoted in a short time, but the decomposition of astaxanthin was also promoted. It was suggested that the shorter the heating time, the more the decomposition of astaxanthin can be suppressed.

(9) Effect of Antioxidants on Cis-Astaxanthin-Containing Composition Table 11 and Figure 11 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at antioxidant concentrations of 0 mM to 20 mM, heating temperature of 160°C, heating time of 5 minutes, and pressure of 10 MPa. The antioxidants did not affect the cis-isomerization ratio of astaxanthin. Ascorbic acid effectively inhibited the decomposition of astaxanthin. BHT slightly inhibited the decomposition of astaxanthin, but α-tocopherol was not confirmed to have an effect of inhibiting the decomposition of ascorbic acid under the above conditions.

(10) Effect of isomerization catalyst on cis-astaxanthin-containing composition Table 12 and Figure 12 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at 0 mM to 100 mM of isomerization catalyst, heating temperatures of 80 ° C. and 120 ° C., heating time of 5 minutes, and pressure of 10 MPa. The addition of the isomerization catalyst promoted the cis-isomerization reaction. Compared to lycopene, the cis-isomerization promotion effect of the isomerization catalyst in astaxanthin was low. The decomposition of lycopene was also promoted by the addition of the isomerization catalyst, but the effect of promoting the decomposition of astaxanthin by the addition of the isomerization catalyst was not confirmed.

(11) Effects of antioxidants and isomerization catalysts in cis-astaxanthin-containing compositions Table 13 and FIG. 13 show the measurement results of the cis-isomerization ratio and carotenoid residual ratio at 0 mM to 10 mM antioxidant (ascorbic acid, VC), 0 mM to 100 mM isomerization catalyst (catalyst), heating temperature 180°C, 200°C, heating time 15 seconds to 60 seconds, and pressure 10 MPa. Under these conditions, the combined effect of the antioxidant and catalyst was not significant, but it was suggested that high temperature and short-time treatment in the presence of an antioxidant (ascorbic acid) was effective. At a heating temperature of 200°C, heating time of 30 seconds, 0 mM catalyst, and 10 mM antioxidant, a cis-isomerization ratio of 60.3% and a carotenoid residual ratio of 93.7% were achieved. FIG. 14 (B) shows a chromatogram of the cis-astaxanthin-containing composition obtained under these conditions.

(12) Effect of heating time and heating temperature on cis-β-carotene-containing composition Tables 14 and 15 show the measurement results of the cis-isomerization ratio and carotenoid residual rate at heating times of 0.5 minutes (30 seconds) to 5 minutes, heating temperatures of 140°C to 200°C, and a pressure of 10 MPa. The higher the heating temperature, the more rapidly cis-isomerization was promoted, but the decomposition of β-carotene was also promoted. It was suggested that the shorter the heating time, the more the decomposition of β-carotene could be suppressed.

(13) Effect of heating time, heating temperature, and antioxidant on cis-β-carotene-containing composition Tables 16 and 17 show the measurement results of the cis-isomerization ratio and carotenoid residual ratio at 0 mg/mL and 1 mg/mL of antioxidant (α-tocopherol), heating time of 0.5 minutes (30 seconds) to 5 minutes, heating temperature of 200°C, and pressure of 10 MPa. It was suggested that high-temperature, short-time treatment in the presence of antioxidant (α-tocopherol) is effective. At a heating temperature of 200°C, heating time of 30 seconds, and antioxidant of 1 mg/mL, a cis-isomerization ratio of 62.0% and a carotenoid residual ratio of 94.9% were achieved.

6. Effects of the Example According to the present example, a method for producing a cis-carotenoid-containing composition can be provided that can efficiently cis-isomerize carotenoid and has a high residual rate of carotenoid after treatment.

The above examples are merely illustrative and should not be construed as limiting the present invention. Although the present invention has been described with reference to exemplary embodiments, the words used in describing and illustrating the present invention are understood to be descriptive and exemplary, rather than limiting. As detailed herein, changes may be made within the scope of the appended claims without departing from the scope or essence of the present invention in its form. Although the present invention has been described herein with reference to specific structures, materials and examples, it is not intended that the present invention be limited to the disclosures therein, but rather that the present invention extends to all functionally equivalent structures, methods and uses within the scope of the appended claims.
The present invention is not limited to the above-described embodiments, and various modifications and variations are possible within the scope of the claims of the present invention.

Claims (6)

A method for producing a cis-carotenoid-containing composition, comprising the steps of:
heating the mixture of trans-carotenoid and organic solvent flowing through the flow path to 120° C. or higher ;
and cooling the heated mixture flowing through the flow path after the heating step,
In the heating step, the organic solvent is in a subcritical fluid state.
A method for producing a cis-carotenoid-containing composition. The method for producing a cis-carotenoid-containing composition according to claim 1, wherein the pressure in the heating step is 5 MPa to 50 MPa. The method for producing a cis-carotenoid-containing composition according to claim 1 or claim 2, wherein the organic solvent is at least one selected from the group consisting of esters having 3 to 6 carbon atoms, alcohols having 1 to 4 carbon atoms, and ketones having 3 to 9 carbon atoms. A method for producing a cis-carotenoid-containing composition according to any one of claims 1 to 3, wherein the heating time in the heating step is 10 minutes or less. The method for producing a cis-carotenoid-containing composition according to any one of claims 1 to 4 , wherein the mixture contains an antioxidant. The method for producing a cis-carotenoid-containing composition according to claim 1 , wherein the mixture contains an isomerization-promoting catalyst.

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