WO2016181719A1 - Method for obtaining purified product of carotenoids - Google Patents

Abstract

The present invention addresses the problem of providing an industrially useful method for contributing to the effective utilization of algal culture residual liquid and producing carotenoids free of hazardous substances. The above problem is solved by a method for obtaining a purified product of carotenoids from a residue from which phycocyanin has been extracted after having produced phycocyanin by culturing algae, the method for obtaining a purified product of carotenoids characterized by having a saponification step for using an alkaline substance to decompose the chlorophyll contained in a carotenoid extract (A) obtained after a step for extracting carotenoids from the residue and filtering, and a carotenoid precipitation step conducted thereafter, the conditions of the saponification step being as follows: (1) 1.9-7.6 molar equivalents of an alkaline substance are used in relation to the solid mass (1 kg) of the carotenoid extract (A); (2) the temperature at which saponification is conducted is 40-80°C.

Description

Method for obtaining a purified carotenoid

The present invention relates to a method for obtaining a purified product of carotenoid, and particularly to a method having a saponification step for decomposing chlorophyll contained in an algal culture and a subsequent carotenoid precipitation step.

Blue-green algae, especially spirulina, are not only used as health foods but also as phycocyanin pigment sources because they contain a large amount of useful substances such as carotenoids, nucleic acid-related substances, amino acids, vitamins, minerals, and phycocyanin pigments. Phycocyanin dyes usually have an absorption maximum wavelength at 618 nm and exhibit a water-soluble and vivid blue color. Therefore, they are used as water-soluble natural colorants for foods such as chewing gum and ice confectionery, or cosmetics such as eye shadows and lipsticks. It is used as a colorant for creams, eyeliners, shampoos, and emulsions.

Carotenoids have an antioxidant effect and are expected to have an antioxidant effect even after being taken into the body, so they are also added to various health foods and supplements. Among carotenoids, zeaxanthin is widely used for the purpose of coloring cultured fish and improving the egg yolk quality of chicken eggs. However, it is only two carotenoids that exist in the macular region of the human retina together with lutein, and it is AMD (aging-related). It is known to be related to a reduction in the risk of macular degeneration (Patent Document 1, Non-Patent Document 1).

Also, zeaxanthin and lutein have been reported to have strong antitumor promoting properties (Non-patent Document 2). Recent reports have shown that it can play an important role in combating conditions that induce cardiovascular disease, atherosclerosis, skin cancer, ovarian cancer, and the like.

The phycocyanin dye can be produced, for example, from the following four steps (Patent Documents 2 and 3).
1) Water extraction process of cyanobacteria algae body components,
2) Centrifugation step,
3) Concentration process by ultrafiltration,
4) Drying process.
However, the residue after separation of phycocyanin generated by this production process (hereinafter referred to as residual liquid) is still disposed of in spite of containing a large amount of nucleic acid-related substances, amino acids, carotenoid pigments, vitamins, minerals, etc. In many cases, this not only becomes a source of environmental pollution, but also leads to the waste of valuable biomass resources. Therefore, effective reuse of the residue is desired.

On the other hand, chlorophyll degradation products are also known to be harmful (Patent Document 4).
Chlorophyll contained in algae can cause photosensitivity, for example, cases of livestock damage due to feeding have been known for a long time, and excessive eating of chlorella tablets is also widely known to cause human injury. It became. This is directly attributed to pheophorbide produced by the decomposition of chlorophyll, and photosensitivity reaction reveals photosensitivity such as sunlight dermatitis, but this pheophorbide used white shark As a result of the phototoxicity test, LD ₅₀ has a value of 45.5 mg / 100 g or more and MLD ₅₀ has a value of 12 mg / 100 g or more (Non-patent Document 3). Moreover, since chlorophyll tends to be acidic or pheophorbide a is easily generated by the coexistence of an organic solvent, there remains a problem in using carotenoids extracted from algae as a health food as they are.

In the purification of carotenoids derived from microalgae, especially zeaxanthin, separation from chlorophyll is difficult and the purification process is complicated. In order to overcome these points, fermentation production by the terrestrial bacterium Erwinia uredovora has been studied, but its yield is extremely low and cannot be practically used (Non-patent Document 4).

JP 2005-536547 A JP-A-1-123865 JP-A-6-16519 Japanese Patent Publication No. 5-27619

J M Seddon et al, Dietary Carotenoids, Vitamins A, C, and E, and Advanced Age-Related Macro Generation, Journal of the American Medical Medical Medical Valminoidｓｓ. 272, no. 9, pages 1413-1420, (1994) L Packer, M Hiramatsu, T Oshikawa, (Editors), Antixidant Food Supplements in Human Health, Academic Press, NY, 1999, Pp223 and Pp226 Journal of Japanese Society of Agricultural Science, 1980, vol54, No9, 721-726 J. et al. Bacteriol. 172, 6704, 1990

As described in the background art, there are no industrially useful production methods that do not contain harmful substances related to carotenoids, particularly zeaxanthin, and production methods for carotenoids that contribute to effective utilization of the residual liquid. There is no current situation.
Therefore, an object of the present invention is to provide an industrially useful production method for carotenoids that contributes to effective utilization of algal culture residue and does not contain harmful substances.
Another object of the present invention is to provide functional foods, feeds, cosmetics, food colors, and the like containing purified carotenoids.

In the present invention, after producing phycocyanin by culturing algae, a method for obtaining a purified product of carotenoid from a residue obtained by extracting the phycocyanin,
A saponification step using an alkaline substance for decomposing chlorophyll contained in the carotenoid extract (A) obtained after the steps of extracting and filtering the carotenoid from the residue, and a subsequent carotenoid precipitation step are provided. And a method for obtaining a purified carotenoid product characterized in that the conditions of the saponification step are as follows.
(1) The alkaline substance is used in an amount corresponding to 1.9 to 7.6 mol of the alkaline substance relative to the solid mass (1 kg) of the carotenoid extract (A).
(2) The temperature for saponification is 40 to 80 ° C.

ADVANTAGE OF THE INVENTION According to this invention, the industrially useful manufacturing method which does not contain the harmful | toxic substance which concerns on carotenoid useful for a living body which contributes to the effective utilization of algae culture residual liquid can be provided.
Furthermore, foods, feeds, cosmetics, or food colors containing purified carotenoids can also be provided.

It is drawing which shows the outline of the process of this invention.

That is, the present invention includes the following items.
1. A method for obtaining a purified product of carotenoid from a residue obtained by extracting phycocyanin after producing phycocyanin by culturing algae,
A saponification step using an alkaline substance for decomposing chlorophyll contained in the carotenoid extract (A) obtained after the steps of extracting and filtering the carotenoid from the residue, and a subsequent carotenoid precipitation step are provided. And a method for obtaining a purified carotenoid product characterized in that the conditions of the saponification step are as follows.
(1) The alkaline substance is used in an amount corresponding to 1.9 to 7.6 mol of the alkaline substance relative to the solid mass (1 kg) of the carotenoid extract (A).
(2) The temperature for saponification is 40 to 80 ° C.
2. 1. the alkaline substance is potassium hydroxide or sodium hydroxide; A method for obtaining a purified product of carotenoid as described in 1.
3. Furthermore, it has a step of extracting carotenoid with ethanol or a water-ethanol mixed solution before the saponification step. Or 2. A method for obtaining a purified product of carotenoid as described in 1.
4). In the precipitation step, carotenoids are precipitated from the liquid obtained by the saponification step by adjusting the water / ethanol mass ratio in the range of 45/55 to 55/45. ~ 3. A method for obtaining a carotenoid purified product according to any one of the above.
5. 1. Algae is a cyanobacterium containing spirulina or phycocyanin ~ 4. A method for obtaining a carotenoid purified product according to any one of the above.
6). 1. The carotenoid is zeaxanthin, β-carotene, or myxoxanthophyll. ~ 5. A method for obtaining a carotenoid purified product according to any one of the above.
7.1. ~ 6. A food, feed, cosmetic, or food color containing carotenoid obtained by the method for obtaining a purified carotenoid product according to any one of the above.

The first step of the present invention is a step of obtaining an extract obtained by extracting phycocyanin in algae into an aqueous suspension. Algae which can be used for the preparation of this extract include the genus Arthrospira, the genus Spirulina, the genus Aphanizomenon, the genus Fischerella, the genus Anabaena, the genus Nesmo (Genus Synechocystis), genus Synechococcus, genus Tolypothrix, genus Aphanothace, genus Mastigoclaus, genus Pleurocapsa To Arsulospira, which has been produced on a scale and has been confirmed to be safe Which is desirable.

Examples of the algae used in the present invention include raw algae and dried algae, but phycocyanin is easily extracted in the step of obtaining an extract obtained by extracting phycocyanin in algae into an aqueous suspension. Dry algae are preferred because the amount of phycocyanin that can be extracted is also stable.

Raw algae are harvested by a method such as centrifugation and filtration of algae cultured in water, and usually contain 70 to 90% by mass of water. Algae are usually cultured in natural water or artificial light in water, but it is preferable to harvest cyanobacteria that are irradiated with light and undergoing photosynthesis. Especially for cyanobacteria grown in outdoor culture tanks under natural light, photosynthesis continues and the water temperature rises after 10 am, compared to cyanobacteria harvested at night or immediately after the start of light irradiation. More preferred are cyanobacteria harvested from sunset to sunset.

Examples of the dried algae include those obtained by freeze-drying or spray-drying raw algae cultured by the above method.

In the method for extracting phycocyanin from algae of the present invention, an extract is obtained in the first step, a calcium salt and phosphate are reacted in the extract in the second step to obtain calcium phosphate, and phycocyanin is added to the calcium phosphate. To adsorb the adsorbed material. In order to perform such an operation in the second step, for example, the first step and the second step may be performed as follows.
1. The first step is a step of preparing an aqueous suspension containing algae and a calcium salt, and obtaining an extract obtained by extracting phycocyanin in the algae into the aqueous suspension. Adding calcium phosphate to obtain calcium phosphate and adsorbing phycocyanin impurities to the calcium phosphate to obtain an adsorbate;
2. The first step is a step of preparing an aqueous suspension containing algae and phosphate, and obtaining an extract obtained by extracting phycocyanin in the algae into the aqueous suspension, and the second step is the extract. Adding calcium salt to obtain calcium phosphate and adsorbing phycocyanin impurities to the calcium phosphate to obtain an adsorbate.
3. In the first step, an extract obtained by extracting phycocyanin in algae into an aqueous suspension is obtained, and in the second step, phosphate and calcium salt are added to the extract to obtain calcium phosphate, and the calcium phosphate is added to the calcium phosphate. A process of obtaining an adsorbate by adsorbing phycocyanin impurities.

In the present invention, the first step is a step of preparing an aqueous suspension containing algae, calcium salt and phosphate, and obtaining an extract obtained by extracting phycocyanin in the algae into the aqueous suspension. Then, the process proceeds to the second step as it is, and calcium phosphate is obtained with the extract, and adsorbate can be obtained by adsorbing phycocyanin impurities to the calcium phosphate. In addition, the 3. In the first step, calcium salt and / or phosphate may be added. In the method for extracting phycocyanin from the algae of the present invention, As a first step, a water suspension containing algae and a calcium salt is prepared as a first step, and a step of obtaining an extract in which phycocyanin in the algae is extracted into the water suspension is obtained. The extraction time of phycocyanin therein can be shortened, and an extract with less elution of phycocyanin impurities, particularly carotenoids, is preferable. The following description of the first step and the second step is 1. The above method is assumed.

Examples of a method for obtaining an extract in the first step include a method of preparing an aqueous suspension containing algae and a calcium salt, and extracting the phycocyanin in the algae by maintaining the extract at 0 to 40 ° C. Is mentioned.

To obtain the aqueous suspension, for example,
First method. Add calcium salt to the algae-suspended aqueous solution.
Second method. Examples include a method of adding algae to an aqueous solution of calcium salt and suspending it, but the second method. Is preferred.

Examples of the calcium salt used in the preparation of the aqueous solution of the calcium salt used in the present invention include water-soluble calcium salts such as calcium chloride, calcium nitrate, and calcium nitrite, among which calcium chloride is preferable.

The concentration of the calcium salt in the aqueous suspension is preferably 0.1 to 10% by mass, more preferably 0.1 to 5% by mass, and still more preferably 0.5 to 3% by mass.

When algae is suspended in an aqueous solution of calcium salt to obtain an aqueous suspension, the aqueous solution of calcium salt is suspended in the range where the concentration of cyanobacteria is 0.1 to 20% by mass in terms of solid content. A range of 2 to 8% by mass is more preferable.

The suspension is preferably prepared in the range where the temperature of the aqueous suspension is 0 to 40 ° C, more preferably 0 to 35 ° C.

An aqueous suspension containing algae and calcium salt is prepared, and phycocyanin in the algae is extracted into the aqueous suspension to obtain an extract. Phycocyanin is extracted from algae by standing still, but may be stirred if necessary. The extraction time is preferably 1 to 48 hours, more preferably 1 to 20 hours.

When obtaining an extract, phycocyanin in algae can be efficiently extracted into an aqueous suspension by adding a basic compound to the aqueous suspension or subjecting it to ultrasonic irradiation. Both the addition of the basic compound and the ultrasonic irradiation treatment may be performed, or only one of them may be performed. When both are performed, the basic compound may be added after ultrasonic irradiation, or the ultrasonic irradiation treatment may be performed after adding the basic compound, but the ultrasonic wave is added after adding the basic compound. It is preferable to perform irradiation treatment.

The irradiation method when performing the ultrasonic irradiation treatment is not limited as long as it can destroy algal cells and promote the transfer of phycocyanin into suspension, and examples include batch type and continuous type. However, a continuous type in which ultrasonic waves are continuously applied is preferable. As a continuous ultrasonic irradiation treatment apparatus, for example, a multiple ultrasonic dispersion apparatus for production manufactured by Nippon Seiki Seisakusho Co., Ltd. may be used.

In the second step, phosphate is added to the aqueous extract of phycocyanin obtained in the first step. The phosphate may be added as a solid or in the form of an aqueous solution. Examples of the phosphate include sodium phosphate such as sodium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate; potassium phosphate such as potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; Examples include magnesium phosphate; water-soluble inorganic salts such as ammonium dihydrogen phosphate, among which sodium phosphate and potassium phosphate are preferable, sodium phosphate is particularly preferable, and disodium hydrogen phosphate is most preferable.

The phosphate is preferably added to the extract so that the phosphate concentration is 1 to 5% by mass in the extract, and is preferably added to the extract so that the concentration is 2 to 3% by mass. Is more preferable.

Add phosphate to the extract. When phosphate is added to the extract, the phosphate reacts with the calcium salt in the water extract to cause precipitation of calcium phosphate and adsorbs and adsorbs chlorophyll and other contaminants contaminated with phycocyanin pigment on the calcium phosphate. Form things. Thereby, the purity of the phycocyanin pigment can be increased. After adding the phosphate, it may be allowed to stand or may be stirred if necessary. The time required for the reaction (adsorption) of calcium ions and phosphate ions is preferably 2 to 10 hours, and more preferably 3 to 5 hours.

The pH when adsorbing phosphate and algae to obtain an adsorbate in the extract is preferably 4 to 8, more preferably 5 to 6, because the amount of phycocyanin obtained is increased. The pH can be adjusted, for example, by adding a basic compound or an acidic compound to the extract. Examples of the basic compound include alkali compounds such as sodium hydroxide, potassium hydroxide and lithium hydroxide; carbonates of alkali metals such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate and lithium carbonate; hydrogen carbonate Examples thereof include alkali metal hydrogen carbonates such as sodium, potassium hydrogen carbonate and lithium hydrogen carbonate; alkali metal acetates such as sodium acetate, potassium acetate and lithium acetate. Examples of the acidic compound include citric acid, hydrochloric acid, lactic acid, acetic acid, and the like. The pH of the extract can also be adjusted by adding a basic compound or acidic compound to the aqueous suspension in advance. If the pH of the aqueous suspension at this time is adjusted to 7 to 6, the pH of the extract is preferably 5 to 6.

In the third step, algal residues and the adsorbate are removed from the extract after completion of the second step, but if the extract contains a chelating agent prior to the third step, the amount of recovered phycocyanin may be increased. Is particularly preferable. This is because phycocyanin includes phycocyanin C and allophycocyanin. Allophycocyanin is adsorbed on calcium phosphate, and allophycocyanin adsorbed on this calcium phosphate is removed together with calcium phosphate in the next third step, but before the third step. The inventor believes that when the chelating agent is contained in the extract, the chelating agent is adsorbed on the calcium phosphate, so that allophycocyanin is separated from the calcium phosphate and remains in the extract.

The chelating agent may be added before the third step. For example, the chelating agent may be added to the algae aqueous suspension in the first step, or may be added to the prepared extract. It may be added to the extract before obtaining the adsorbate in the process, or may be added after obtaining the adsorbate. In the present invention, it is preferably added at the time of suspension adjustment.

Examples of the chelating agent include organic carboxylates such as sodium citrate, sodium oxalate, sodium tartrate, sodium gluconate; nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminopentaacetic acid (DTPA) Amino carbonates such as dihydroxyethyl glycine (DFG), triethanolamine (TEA), N- (2-hydroxyethyl) iminodiacetic acid (HEIDA), hydroxyethylenediaminetetraacetic acid (HEDTA) and the like; Examples thereof include ether carboxylates such as sodium carboxymethyltaltronate (CMT) and sodium carboxymethyloxysuccinate (CMOS). Of these, sodium citrate and sodium ethylenediaminetetraacetate are more preferable.

The amount of chelating agent added is preferably 5 to 100% by mass, more preferably 10 to 40% by mass based on the amount of calcium chloride used.

Algal residue can be obtained from the extract in the third step. Examples of means for obtaining these include various methods, such as a filtration method using a filter medium such as filter paper and filter cloth, a decantation method performed by collecting the supernatant from the precipitate, and a centrifugation method. Of these, separation by centrifugation is preferable.

Centrifugation may be performed under conditions that can remove algae residues and adsorbates from the extract, but is preferably performed under a centrifugal acceleration of 1,000 to 30,000 G for 10 seconds to 2 hours, and a gravitational acceleration of 3 Centrifugation conditions of 1 to 30 minutes at 1,000 to 10,000 G are more preferred. There are disperse type centrifuges, alpha type centrifuges, and shear press type centrifuges as centrifuges. However, since the workability is improved, the disperse type centrifuges and alpha centrifuges are improved. Combination continuous centrifugation is preferred.
In addition, the obtained residue is preferably dried by a spray dryer method, a freeze-drying method or the like in order to prevent deterioration of the carotenoids contained, particularly zeaxanthin.

Next, a step of extracting carotenoid from the residue obtained by the above operation is performed.
This extraction method can be performed by a known and commonly used method. For example, it is preferable to perform a solvent extraction method using ethanol or a water-ethanol mixed solution as an extraction solvent.
Furthermore, when a water-ethanol mixed solution is used, it is preferably 99 wt% (mass%) to 80 wt% (mass%) in order to increase the extraction selectivity of zeaxanthin. If it is higher than the above range, ethanol and lipid will undergo transesterification to produce unpurified products such as fatty acid ethyl ester, and if it is lower, the zeaxanthin extraction efficiency will be reduced.
The temperature at the time of extraction can be in the range of 20 to 80 ° C., but in order to perform the extraction in a short time, it is preferable to carry out at 40 to 80 ° C. while stirring the solution uniformly. Is preferably extracted in a temperature range of 50 to 80 ° C.

Next, the process of filtering the extract obtained by said operation is performed.
This step can be performed by a known and commonly used method. For example, it can be filtered using a commercially available filter paper.

Next, the extract is concentrated to obtain a carotenoid extract (A).
The solid component mass of the extract (A) is preferably 3.3 wt% to 33 wt% of the extract (A), more preferably 3.3 wt% to 13 wt%, and 5 wt% to 8.3 wt%. Particularly preferred.
Here, the solid content mass is measured after drying the solvent at 105 ° C. after distilling off the solvent at a vacuum of 10 kPa or less while heating a part of the obtained extract at 40 ° C.

Next, the saponification process of the obtained carotenoid extract (A) is performed. The purpose of this step is to remove chlorophyll contained in the extract (A).
In the saponification step of the present invention, it is preferable to treat the alkaline substance in an amount corresponding to 1.9 to 7.6 mol equivalent to the solid mass (1 kg) of the carotenoid extract (A). Furthermore, a treatment with an amount corresponding to 3.8 mol is particularly preferred.
The alkaline substance used in the present invention can be used without limitation as long as it is usually an alkaline substance capable of removing chlorophyll, but preferably potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, etc. Mention may be made of alkaline substances.

As the mass of the alkaline substance to be used, a numerical mass converted to a substantial amount of the alkaline substance is used. For example, when potassium hydroxide is used as the alkaline substance, since commercially available potassium hydroxide has a content of about 85%, the mass of the numerical value converted from this is used.
When the added amount of the alkaline substance is used, the intended saponification can be performed in a short time at 30 ° C. or higher. To sufficiently saponify the chlorophyll, the solution is stirred so as to be uniform. However, it is preferably carried out at 40 to 80 ° C.

The time required for saponification is usually 30 minutes to 24 hours. When the time is shorter than this time, saponification of chlorophyll is insufficient, and when the time is longer, carotenoid is deteriorated.

Next, a precipitation step for obtaining a purified carotenoid product is performed.
In this step, it is preferable to adjust the solvent concentration at the time of carotenoid precipitation, and it is preferable to add water to adjust the concentration of the precipitate washing solution.
More specifically, carotenoids are precipitated by adding water to the solution obtained after the saponification step and adjusting the water-ethanol concentration. The water-ethanol concentration is preferably 70 wt% or less, and more preferably in the range of 45 wt% to 55 wt% when it is precipitated in a short time.

In the present invention, diatomaceous earth filtration, diatomaceous earth washing, and diatomaceous earth elution may be performed.
That is, in this step, for example, a solution in which carotenoids are precipitated with 45 wt% ethanol water is filtered through diatomaceous earth, and then the filtrate is washed with 45 wt% ethanol water to elute impurities.
Subsequently, the carotenoid eluate can be purified by eluting the filtrate with 99 wt% to 80 wt% ethanol water.
This step has an effect that the purified carotenoid eluate is rich in zeaxanthin.

The carotenoid obtained from the present invention can be used as a functional product in foods, feeds, cosmetics, food colors, etc. and used for various applications.
Examples of methods for producing foods, feeds, cosmetics, edible pigments and the like as the functional products include publicly known methods.

Hereinafter, the present invention will be described more specifically with reference to measurement methods according to the present invention, examples, and comparative examples.
<Measurement of zeaxanthin concentration>
The concentration of zeaxanthin in the extract, saponified extract, and final purified product was determined by replacing the solution with methyl tert-butyl ether: acetonitrile = 1: 1 (volume ratio), followed by filtration with a 0.2 μm syringe filter, and UPLC (WACQUITY UPLC; Waters) can be quantified under the following conditions.
Specifically, Acquity UPLC HSS T3 (φ2.1 mm × 100 mm, 1.8 μm) was used for the column, and acetonitrile: methanol: methyl tert-butyl ether = 70: 20: 10 (volume ratio) as the solvent 1 for the mobile phase. A 10 mM ammonium acetate aqueous solution is used as the solvent 2, the concentration gradient of the solvent 1 is 60% -4 min-75% -10 min-100% -10 min-98% -1 min-60% (3 min), and the flow rate is 0.3 to 0.35 mL. It can be measured by setting a condition of a column oven temperature of 40 ° C.

<Measurement of zeaxanthin amount in each purification step>
(1) A normal hexane: acetone: ethanol: toluene = 10: 7: 6: 7 (volume ratio) mixed solvent is added to 0.2 g of the phycocyanin extraction residue, and the mixture is extracted for 30 minutes while refluxing. After the volume of the extract is changed, the solvent is replaced with methyl tert-butyl ether: acetonitrile = 1: 1 (volume ratio), and measurement is performed by UPLC, whereby the amount of zeaxanthin in the phycocyanin extraction residue can be measured. This measured value is assumed to be Z1.
(2) A part of the phycocyanin extraction residue was extracted from the extract so that the amount was 0.2 g, and the solvent was replaced with methyl tert-butyl ether: acetonitrile = 1: 1 (volume ratio), and quantitative measurement was performed with UPLC. By performing, the amount of zeaxanthin in the extract can be measured. This measured value is set as Z2. The amount of zeaxanthin can be measured in the same manner for the saponified solution and the final purified product. The amount of zeaxanthin in the saponified solution is Z3, and the amount of zeaxanthin in the final purified product is Z4.

<Zeaxanthin recovery rate calculation method>
By using the amount of zeaxanthin in each solution obtained, the zeaxanthin recovery rate in each step can be calculated. That is, the extraction process recovery rate can be calculated as Z2 / Z1 × 100%, the saponification process recovery rate can be calculated as Z3 / Z2 × 100%, and the purification process recovery rate can be calculated as Z4 / Z3 × 100%.

2) Content of zeaxanthin in the final purified product The final purified product is dried with a dryer at 105 ° C., and the solid content mass is measured. The value obtained by dividing the mass of zeaxanthin by the mass of solid content was defined as the zeaxanthin content of the final product.

3) Chlorophyll content The solvent of the sample solution is replaced with 85% aqueous acetone. Filter through a 3G2 glass filter and wash the filter residue with 85% acetone. Diethyl ether (ether) and water are added to the filtrate in a ratio of 1: 1 and distributed, and the ether layer is separated. Half of the ether is added to the aqueous layer and water is added in the same amount as the ether, and the resulting ether layer is added to the initially distributed ether layer. An equal amount of water is added to the combined ether layers and distributed to obtain an ether layer. The operation of adding an ether layer to obtain an ether layer is repeated three times, and the absorbance of the finally obtained ether layer at 642.5 nm and 660 nm is measured to obtain the total chlorophyll content in the sample solution.

(Production Example 1)
<Adjustment of phycocyanin residue>
Using artificial light, Spirulina was cultured and harvested in a culture tank under continuous light irradiation for 7 days and harvested. 1 kg of the obtained raw algal bodies (solid content: 18.0%) is added to 6 L (0.09 mol / liter) of 1% calcium chloride solution and stirred at 20 ° C. for 45 minutes to obtain a spirulina suspension. It was. 3 g of sodium carbonate and 3 g of sodium hydrogen carbonate were dissolved in 50 mL of water and added to the Spirulina suspension (the concentration of the basic compound in the suspension was 0.01 mol / liter). The suspension was stirred for 2 minutes and then allowed to stand at 20 ° C. for 5 hours. This suspension was introduced into a continuous ultrasonic crusher (Nippon Seiki Seisakusho RUS-300TCVP type, frequency 19.5 kHz, ultrasonic irradiation unit volume 10 mL, output 300 W), and the suspension was 5 mL / second. Ultrasonic treatment was performed at a speed to the ultrasonic irradiation unit to obtain an extract. To the obtained extract, sodium dihydrogen phosphate was added to a concentration of 6.8 g / liter and disodium hydrogen phosphate to a concentration of 2.2 g / liter. After stirring, the mixture was introduced into a centrifuge and the gravitational acceleration was 10 Centrifugation was performed at 1,000 G for 15 minutes, and the phycocyanin residue and the phycocyanin solution were separated by filtration. Further, the phycocyanin residue was dried by a spray dryer method or the like to obtain a dried phycocyanin residue (blue residue).

(Example 1)
Zeaxanthin was extracted while stirring 300 g of the blue extraction residue in 900 g of a 95 wt% aqueous ethanol solution at 50 ° C. for 1 hour at 300 rpm. Solid-liquid separation was performed by filtration to obtain a filtrate and a filtration residue. The filtration residue was extracted again with a 95 wt% aqueous ethanol solution and added to the filtrate to obtain an extraction solution. The extract was concentrated under reduced pressure to 450 g using a rotary evaporator, and 30 g of a 25% KOH / EtOH solution (an equivalent amount of 3.8 mol of an alkaline substance was used relative to the solid mass (1 kg)) was added. Saponification was performed while stirring at 200 rpm at 50 ° C. for 1 hour to obtain a saponified extract having saponified chlorophyll water-solubilized.

Water was added to the saponified extract to adjust the water ethanol concentration to 45 wt%. The mixture was allowed to stand at 4 ° C. for 30 minutes or more to precipitate a red substance. The saponified extract having the precipitate was passed through diatomaceous earth pre-coated to a thickness of 10 mm, and the precipitate and the liquid were separated by filtration and then washed with a 45 wt% aqueous ethanol solution to remove impurities. The precipitate from which impurities were removed was dissolved in a 95 wt% aqueous ethanol solution to obtain a final purified product.

The extraction residue as a raw material and the extract, saponified extract, and final purified product obtained above were measured by UPLC, and each zeaxanthin recovery rate was calculated. In the saponified extract, the chlorophyll content was calculated. Furthermore, in the final purified product, the dry mass was measured, and the zeaxanthin content in the solid content was calculated. The measurement results were 80% zeaxanthin recovery, 13% zeaxanthin content, and 0.1% or less chlorophyll content.

(Example 2)
150 g of a 95 wt% aqueous ethanol solution was added to 50 g of the blue residue, and extracted with stirring at 78 ° C. for 1 hour. Solid-liquid separation was performed by filtration to obtain a filtrate and a filtration residue. The filtration residue was extracted again with a 95 wt% aqueous ethanol solution and added to the filtrate to obtain an extraction solution. A 95 wt% aqueous ethanol solution was added to the extract to make 500 g. 50 g was collected, and 0.25 g of 25% KOH / EtOH was added (the amount corresponding to 1.9 mol of the alkaline substance relative to the solid mass (1 kg) was used), and saponified while stirring at 78 ° C. for 1 hour. The saponified extract had a chlorophyll content of 0.1% or less and a zeaxanthin recovery rate of 85%.

(Example 3)
150 g of a 95 wt% aqueous ethanol solution was added to 50 g of the blue residue, and extracted with stirring at 78 ° C. for 1 hour. Solid-liquid separation was performed by filtration to obtain a filtrate and a filtration residue. The filtration residue was extracted again with a 95 wt% aqueous ethanol solution and added to the filtrate to obtain an extraction solution. A 95 wt% aqueous ethanol solution was added to the extract to make 500 g. 50 g was collected, and 1.0 g of 25% KOH / EtOH was added (alkaline substance equivalent to 7.6 mol was used with respect to the solid mass (1 kg)), and saponified while stirring at 78 ° C. for 1 hour. The saponified extract had a chlorophyll content of 0.01% and a zeaxanthin recovery rate of 77%.

(Comparative Example 1)
47 kg of an acetone / ethanol (2: 1) mixed solvent was added to 20 kg of the blue residue, and the mixture was extracted at 60 ° C. for 2 hours with stirring at 80 rpm. Solid-liquid separation was performed by filtration to obtain a filtrate and a filtration residue. The filtration residue was extracted again with the mixed solvent and added to the filtrate to obtain an extraction solution. The extract had a zeaxanthin recovery rate of 85%, a zeaxanthin content of 0.8%, and a chlorophyll content of 5.9%.

(Comparative Example 2)
90 g of a 95 wt% aqueous ethanol solution was added to 30 g of the blue residue, and extracted with stirring at 78 ° C. for 1 hour. Solid-liquid separation was performed by filtration to obtain a filtrate and a filtration residue. The filtration residue was extracted again with a 95 wt% aqueous ethanol solution and added to the filtrate to obtain an extraction solution. The extract was concentrated to 90 g. 15 g was collected, 0.5 g of 25% KOH / EtOH was added (3.8 mol equivalent of alkaline substance to the solid mass (1 kg)), and saponified while stirring at 30 ° C. for 1 hour. The saponified extract had a chlorophyll content of 0.01% and a zeaxanthin recovery rate of 80%. However, thin-layer chromatography confirmed that many substances that appeared to be chlorophyll degradation products remained.

(Comparative Example 3)
150 g of a 95 wt% aqueous ethanol solution was added to 50 g of the blue residue, and extracted with stirring at 78 ° C. for 1 hour. A 95 wt% aqueous ethanol solution was added to the extract to make 500 g. 50 g was collected, and 0.5 g of 25% KOH / EtOH was added (3.8 mol of an alkaline substance was used with respect to the solid mass (1 kg)), and saponified with stirring at 78 ° C. for 1 hour. The saponified solution was separated into solid and liquid by filtration to obtain a filtrate and a filtration residue. The filtration residue was extracted again with a 95 wt% aqueous ethanol solution and added to the filtrate to obtain an extraction saponification solution. The saponified extract had a chlorophyll content of 0.83% and a zeaxanthin recovery rate of 84%.

From the above Examples and Comparative Examples, it is clear that a purified carotenoid with a high amount of zeaxanthin and a low content of impurities can be obtained by the method having the requirements constituting the present invention.

The purified product such as carotenoid obtained in the present invention can be used as, for example, functional foods, feeds, cosmetics, food colors, etc. contained in foods, feeds, cosmetics, food colors and the like.

Claims (7)

A method for obtaining a purified product of carotenoid from a residue obtained by extracting phycocyanin after producing phycocyanin by culturing algae,
A saponification step using an alkaline substance for decomposing chlorophyll contained in the carotenoid extract (A) obtained after the steps of extracting and filtering the carotenoid from the residue, and a subsequent carotenoid precipitation step are provided. And a method for obtaining a purified carotenoid product characterized in that the conditions of the saponification step are as follows.
(1) The alkaline substance is used in an amount corresponding to 1.9 to 7.6 mol of the alkaline substance relative to the solid mass (1 kg) of the carotenoid extract (A).
(2) The temperature for saponification is 40 to 80 ° C. The method for obtaining a purified product of carotenoid according to claim 1, wherein the alkaline substance is potassium hydroxide or sodium hydroxide. The method for obtaining a purified product of carotenoid according to claim 1 or 2, further comprising a step of extracting carotenoid with ethanol or a water-ethanol mixed solution and filtering before the saponification step. The carotenoid is precipitated from the liquid obtained by the saponification step by adjusting the mass ratio of water / ethanol in the range of 45/55 to 55/45 in the precipitation step. A method for obtaining a purified carotenoid. The method for obtaining a purified product of carotenoid according to any one of claims 1 to 4, wherein the algae is spirulina or a cyanobacteria containing phycocyanin. 6. The method for obtaining a purified product of carotenoid according to claim 1, wherein the carotenoid is zeaxanthin, β-carotene, or myxoxanthophyll. A food, feed, cosmetic, or food color containing carotenoid obtained by the method for obtaining a carotenoid purified product according to any one of claims 1 to 6.

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