JP5471824B2 - Method for producing carotenoid composition, method for producing high concentration carotenoid composition, method for producing high purity free carotenoid composition, carotenoid composition, high concentration carotenoid composition, and high purity free carotenoid composition - Google Patents

Description

  The present invention relates to a method for producing a carotenoid composition, a method for producing a high concentration carotenoid composition, a method for producing a high purity free carotenoid composition, a carotenoid composition, a high concentration carotenoid composition, and a high purity free carotenoid composition. In particular, a method for producing a carotenoid composition used for pharmaceuticals, quasi drugs, supplements, or food additives, a method for producing a high-concentration carotenoid composition, a method for producing a high-purity free carotenoid composition, a carotenoid composition The present invention relates to a high-concentration carotenoid composition and a high-purity free carotenoid composition.

Carotenoids are a type of pigment component produced by animals, plants, and microorganisms.
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. In fact, the results of epidemiological studies using these carotenoid intakes and blood concentrations as indicators strongly suggest a link between carotenoid intake and disease prevention.

Among the carotenoids, six kinds of β-carotene, lycopene, lutein, zeaxanthin, α-carotene, and β-cryptoxanthin are known as main carotenoids derived from vegetable foods.
These six types of carotenoids are characterized by the composition of carotenoids contained in foods and plants.
For example, β-carotene is abundant in green and yellow vegetables such as carrots and pumpkins, and is also contained in fruits such as mango. Also, lycopene is abundantly contained in tomatoes. In addition, lutein is contained in egg yolk and green-yellow vegetables, and marigold flowers are used as an extraction raw material. Zeaxanthin is contained in corn seed and egg yolk. Further, α-carotene is contained in greenish yellow vegetables.
These food-derived carotenoids are ingested in the food chain of animals and plants including humans, and no problem has been reported regarding safety when ingested as food.
For this reason, colorants such as carrot carotene, orange pigment, tomato pigment, corn pigment and marigold pigment obtained by solvent extraction of carotenoids from these materials are distributed as food additives.

By the way, β-cryptoxanthin is one of the main food-derived carotenoids detected in human blood. β-cryptoxanthin is mainly included in mandarinoids such as Citrus unshiu Marc., Citrus tangerina Hort. ex Tanaka, and Ponkan (Citrus reticulata Blanco). In addition, as other foods, oysters (Diospyros kaki Thunb.), Loquat (Eriobotrya japonica (Thunb.) Lindl.) And the like are also included in papaya (Carica papaya L.) as a main carotenoid in tropical fruits. The amount contained in other foods is small.
Of these, the β-cryptoxanthin content in Satsuma mandarin pulp is as high as 1 to 2 mg per 100 g. For this reason, it is known that Japanese people who eat Eunshu mikan often have a higher β-cryptoxanthin concentration in blood than in other countries.
From a nutritional point of view, β-carotene and β-cryptoxanthin function as provitamin A. Since carotenoids exhibit yellow to red color, they are used as coloring agents for foods and drinks and pharmaceuticals.
β-cryptoxanthin has been suggested by epidemiological studies to have effects such as alcoholic liver injury, osteoporosis, diabetes, rheumatism, and prevention of arteriosclerosis. At the animal level, effects such as carcinogenesis suppression and periodontal disease prevention have been confirmed. Thus, β-cryptoxanthin is expected to have a preventive effect on lifestyle-related diseases, and has many effects that are not observed with other carotenoids but only with β-cryptoxanthin.
For this reason, a high concentration β-cryptoxanthin composition suitable for use in pharmaceuticals, quasi drugs, functional foods, and other foods is highly desired. In other words, although the addition of β-cryptoxanthin to complex carotenoid supplements has been studied, unlike other carotenoids, a means for supplying large quantities has not been established and commercialization has not progressed, so it is not available in the market. was difficult.

Here, a conventional carotenoid extraction and production method will be described.
Conventional carotenoid production methods include synthesis by chemical conversion from other carotenoids, biological synthesis by microorganisms, extraction from plant bodies, and the like.
For example, an extraction method using an organic solvent is known as a method for extracting a carotenoid from a plant body. Examples of carotenoids extracted from plants such as vegetables and fruits with organic solvents are carrot carotene, orange pigment, tomato pigment, corn pigment, and marigold pigment.

Among these, as a conventional method for producing β-cryptoxanthin, methods such as synthesis by chemical conversion from other carotenoids, biological synthesis by microorganisms, extraction from plants such as citrus and oysters are known. Yes.
Here, referring to Patent Document 1, a method for obtaining β-cryptoxanthin from lutein through a chemical reaction is described (Japanese Patent Publication No. 2007-509957). However, there is a problem that high purity β-cryptoxanthin cannot be obtained unless the raw material lutein is purified. In addition, the production method by synthesis of β-cryptoxanthin is easy to scale up, but there is a problem of processing by-products accompanying the synthesis, and consumer psychological resistance due to not being naturally derived β-cryptoxanthin. There was a problem that there was.
Further, in biological synthesis, β-cryptoxanthin is obtained as a by-product in the reaction, so there is a problem that it is expensive to produce only β-cryptoxanthin.
Further, when β-cryptoxanthin is extracted from citrus fruits and oyster fruits by a normal carotenoid extraction method, there is a problem that the concentration is lowered.
Such a low-concentration composition has no problem if it is used for coloring purposes, but for example, when considering use in supplements, an extract with a higher concentration is required to obtain the required supplement content. Therefore, it was not realistic.

On the other hand, in the squeezing process of mandarin oranges, the peels and carcass are removed, and dietary fiber (pulp) is removed by operations such as pulper, finisher, and centrifugal separation to further increase the commercial value of fruit juice. . In addition, the skin generated when making dried straw is often discarded.
The peel and pulp are sometimes used as livestock feed and food additives, but many have been discarded. In particular, most of the residues generated in the squeezing process when processing mandarin oranges with a large amount of production are discarded and not effectively used.
Here, as described above, β-cryptoxanthin is contained in mandarin oranges and oysters, but is also contained in these discarded portions and is promising as an extraction raw material for β-cryptoxanthin.
In fact, a technique relating to a method for producing a β-cryptoxanthin composition by centrifugal concentration and solvent extraction using pulp produced in the squeezing process of Citrus unshiu as a raw material has also been developed.
However, the composition obtained by the method for producing these β-cryptoxanthin compositions has a low concentration of β-cryptoxanthin, and there has been no actual use example.
For this reason, the technique of utilizing these residues as a feedstock of the density | concentration (beta) cryptoxanthin composition which can be utilized for a supplement is calculated | required.

Below, the conventional method of extracting (beta) -cryptoxanthin using the residue which generate | occur | produces in a fruit skin or a squeezing process is demonstrated.
With reference to Patent Document 2, a technique relating to the concentration of β-cryptoxanthin is described (Japanese Patent Laid-Open No. 2000-23637). However, as an example of Patent Document 2, in the method of obtaining a high-concentration fraction from Satsuma mandarin pulp by centrifugal separation, the concentration of β-cryptoxanthin was as low as about 0.67%.
If patent document 3 is referred, the extract obtained by ethanol extraction and water washing | cleaning from the juice residue of Satsuma mandarin is described (Unexamined-Japanese-Patent No. 2008-297215). However, the concentration of β-cryptoxanthin obtained by this method was also as low as 4%.
With reference to Patent Document 4, a method for producing high-purity β-cryptoxanthin using an advanced chromatographic method such as a high performance liquid chromatograph is described (see Japanese Patent Application Laid-Open No. 2000-136181). In the method of Patent Document 4, β-cryptoxanthin with high purity can be obtained, but complicated processing is required to separate the hydrolyzed free carotenoid, and β-crypt that is originally contained in the raw material itself. The cost was high due to the low concentration of xanthine. Furthermore, the amount of organic solvents that are difficult to use in foods such as acetonitrile was also large.
With reference to Patent Document 5, a technique for producing an extract containing a β-cryptoxanthin component using oyster peel that is known to contain a relatively high concentration of β-cryptoxanthin is described (patent document 5). (See Kai 2004-331528). In this method, the fruit skin is extracted with ethanol and concentrated under reduced pressure, and the low boiling point components are removed during the concentration process under reduced pressure, whereby the characteristic dried odor can be reduced. However, this production method is not intended to produce a high concentration of β-cryptoxanthin, and the concentration thereof remains at 0.01% (1 mg / 100 mL) or less.
Referring to Patent Document 6, a low-concentration β-cryptoxanthin composition is similarly described (see JP 2004-329058 A). Although this composition shows a certain improvement in the color tone improvement of Valencia juice that does not contain β-cryptoxanthin at all, it is expected that it is difficult to maintain a constant concentration by adding it to a supplement or the like. The
With reference to Patent Document 7, a method of increasing the extraction efficiency by drying oyster peel before extraction has also been studied (see JP 2009-0500188 A). However, even with this method, the concentration could not be increased.

Referring to Patent Document 8, as a method for producing carotene (β-carotene) from a carotene-containing plant using an organic solvent, β-carotene is extracted from carrot using hexane, and the concentrated solution is cooled to −20 ° C. A method for obtaining a precipitate containing 50 to 60% β-carotene has been shown (see JP-A-4-95066). However, carrot has a carotenoid content of about 9 mg per 100 g of fresh weight (Five Food Composition Table), which is higher than other foods. Carrot carotenoids are α-carotene and β-carotene, 90% of which are hydrocarbon-based carotenoids, and 109 mg of this β-carotene can be dissolved in 100 mL of hexane at 0 ° C. For this reason, it is considered that β-carotene was precipitated by cooling the concentrate to −20 ° C.
With reference to Patent Document 9, a method for obtaining a composition containing carotenoids at a high concentration from marigold petals is described (see Japanese Patent Application Publication No. 2008-538697). However, in the composition obtained by this method, zeaxanthin was the main component, and it was less than 1% of that containing β-cryptoxanthin.
Thus, according to the above-mentioned examples of Satsuma mandarin and oysters, zeaxanthin, lutein, and β-cryptoxanthin, which are ester-type xanthophylls, are soluble in hexane, and a high concentration in the precipitation method by simply cooling the concentrated solution. It is clear that it is difficult to make it.

As described above, high concentration is essential for general-purpose use such as supplements using β-cryptoxanthin. However, in the example in which β-cryptoxanthin was increased in concentration by simple solvent extraction, the concentration was insufficient for the above utilization.
Even when a pure β-cryptoxanthin is produced by incorporating a separation method by a high performance liquid chromatographic method, the cost is enormous even if an Satsuma mandarin byproduct having a high β-cryptoxanthin content is used. Furthermore, in the technique of Patent Document 4, when a large amount is required, it takes a long time to prepare (see Japanese Patent Application Laid-Open No. 2000-136181).

Special table 2007-509957 JP 2000-23637 A JP 2008-297215 A JP 2000-136181 A JP 2004-331528 A JP 2004-329058 A JP 2009-050188 A Japanese Patent Laid-Open No. 4-95066 Special table 2008-538697 gazette

As described above, to produce a carotenoid composition containing a concentration of β-cryptoxanthin that can be used for pharmaceuticals, quasi drugs, supplements, or food additives, costs such as advanced chromatographic methods are required. There was a problem that this method could only be used.
For this reason, the method of manufacturing the carotenoid composition containing (beta) -cryptoxanthin of the density | concentration which can be utilized for a pharmaceutical, a quasi-drug, a supplement, or a food additive at low cost was calculated | required.

  This invention is made | formed in view of such a condition, and makes it a subject to eliminate the above-mentioned subject.

The method for producing a carotenoid composition of the present invention obtains a hexane extract in a soluble part by adding hexane to a powder obtained by drying the water content of a plant containing carotenoid to 5% or less,
Concentrate the hexane extract to obtain a concentrate, add a redissolved solution to the concentrate to redissolve it, cool to about -20 ° C to -30 ° C, and then centrifuge to recover the soluble part. To obtain a low-concentration carotenoid composition, add a predetermined amount of ethanol to the low-concentration carotenoid composition, cool to about -20 ° C to -30 ° C, and centrifuge to obtain an insoluble part , The plant body is one of mandarin oranges, oysters (Diospyros kaki Thunb.), Loquat (Eriobotrya japonica (Thunb.) Lindl.), Or papaya (Carica papaya L.) .
In the method for producing a carotenoid composition of the present invention, the powder is obtained by drying the peel of citrus fruits, and the concentrate is obtained by adding 10% to 20% ethanol to the hexane extract by about 5 times its weight. %: A residue obtained by adding 80 to 90% of water and distilling under reduced pressure while heating at 50 ° C to 60 ° C. The redissolved solution added to the concentrate is 3 times the weight of the concentrate. The predetermined amount of ethanol added to the low-concentration carotenoid composition is about 3 to 4 times the weight of the low-concentration carotenoid composition.
In the method for producing a carotenoid composition according to the present invention, the powder is a powder obtained from a pulp of a site originating from citrus sand, and the redissolved solution added to the concentrate has a weight of the concentrate. It is about 3 times hexane, and the predetermined amount of ethanol added to the low-concentration carotenoid composition is about three times the weight of the low-concentration carotenoid composition.
In the method for producing a carotenoid composition of the present invention, the powder is a powder obtained from a pulp obtained by recovering diatomaceous earth adsorbing insoluble matter generated in a filtration process step during the production of citrus clear fruit juice, The redissolved solution added to the concentrate is about three times the weight of the concentrate, and the predetermined amount of ethanol added to the low-concentration carotenoid composition is about three times the weight of the low-concentration carotenoid composition. It is characterized by being ethanol.
In the method for producing a high-concentration carotenoid composition of the present invention, a hexane / ethanol mixed solution containing 40% hexane and 60% ethanol in a volume ratio is added to the carotenoid composition in an amount of about 3 to 4 times. It is cooled to -30 ° C., centrifuged, and the insoluble part is recovered and concentrated under reduced pressure.
In the method for producing a high-concentration carotenoid composition of the present invention, a hexane / ethanol mixed solution containing 40% hexane and 60% ethanol in a volume ratio is added to the carotenoid composition in an amount of about 3 to 4 times. It is cooled to −30 ° C., centrifuged, and the insoluble part is recovered.
The method for producing a high-purity free carotenoid composition of the present invention comprises hydrolyzing the high-concentration carotenoid composition, removing alkali by washing with water, adding a predetermined amount of hexane to the residue obtained by concentrating the organic phase, The insoluble part obtained by dispersing and leaving by sonication is collected by filtration, ethanol is added to the insoluble part and dispersed by sonication, and the insoluble part produced by standing is recovered by filtration. .
The method for producing a high-purity free carotenoid composition of the present invention comprises hydrolyzing the high-concentration carotenoid composition, removing alkali by washing with water, adding a predetermined amount of hexane to the residue obtained by concentrating the organic phase, The insoluble part obtained by being allowed to stand after being dispersed by sonication is collected by filtration, ethanol is added to the insoluble part and dispersed by sonication, and the insoluble part resulting from standing is recovered by filtration. And
In the method for producing a high purity free carotenoid composition of the present invention, the high-concentration carotenoid composition is hydrolyzed and the alkali is removed by washing with water, and then the hexane phase and the dark red intermediate phase are recovered and concentrated. % To 95%: suspended in a hexane / acetone mixed solution containing 5% to 15% acetone, centrifuged to collect the supernatant, and the supernatant was recovered by column chromatography using silica gel to separate the hexane / acetone. Elution is performed using a mixed solution, and the thickest red band portion is fractionated and collected.
In the method for producing a high-concentration carotenoid composition of the present invention, a hexane / ethanol mixed solution containing 40% hexane and 60% ethanol in a volume ratio is added to the carotenoid composition in an amount of about 3 to 4 times. The solution is cooled to −30 ° C., centrifuged, the soluble part is recovered, and the powder is obtained by drying oyster skin .
The method for producing a high-purity free carotenoid composition of the present invention comprises hydrolyzing the high-concentration carotenoid composition, removing alkali by washing with water, adding a predetermined amount of hexane to the residue obtained by concentrating the organic phase, The insoluble part obtained by dispersing and leaving by sonication is collected by filtration, ethanol is added to the insoluble part and dispersed by sonication, and the insoluble part produced by standing is recovered by filtration. .

  According to the present invention, it is possible to provide a carotenoid production method capable of producing a carotenoid at a concentration that can be used for pharmaceuticals, quasi drugs, supplements, or food additives by extraction with hexane and ethanol. it can.

It is a flowchart of the extraction method of (beta) -cryptoxanthin which concerns on embodiment of this invention. It is a graph which shows the extract liquid composition which concerns on carotenoid extraction which concerns on embodiment of this invention, and the distribution amount of collection | recovery. It is a graph which shows the composition of the redissolved solution which concerns on embodiment of this invention, and distribution to a soluble part and an insoluble part. It is a graph which shows the ethanol content and the distribution of (beta) -cryptoxanthin in the redissolved solution which concerns on embodiment of this invention. It is a graph which shows the cooling temperature at the time of hexane remelting which concerns on embodiment of this invention, and the weight of a soluble part and an insoluble part. It is a graph which shows the (beta) -cryptoxanthin content in the soluble part and insoluble part at the time of hexane redissolving which concerns on embodiment of this invention. It is a graph which shows (beta) -cryptoxanthin content rate (%) in the soluble part and insoluble part at the time of hexane redissolving which concerns on embodiment of this invention. It is a graph which shows the weight ratio (%) of the cooling temperature at the time of ethanol redissolving which concerns on embodiment of this invention, and a soluble part and an insoluble part. It is a graph which shows (beta) -cryptoxanthin content (mg / 100g) in the soluble part at the time of ethanol redissolving which concerns on embodiment of this invention, and an insoluble part. It is a graph which shows (beta) -cryptoxanthin content rate (%) in the soluble part at the time of ethanol redissolving which concerns on embodiment of this invention, and an insoluble part. It is a graph which shows the mix ratio of the hexane / ethanol solution which concerns on embodiment of this invention, and (beta) -cryptoxanthin content rate (%) in a soluble part and an insoluble part. It is a figure which shows the yield of the (beta) cryptoxanthin concentrated composition in the centrifugation pulp which concerns on embodiment of this invention, a content, content rate, and a recovery rate. It is a figure which shows the yield of the (beta) cryptoxanthin concentrated composition in the filtration treatment diatomaceous earth which concerns on embodiment of this invention, a content, content rate, and a recovery rate. It is a graph which shows the result of HPLC (high performance liquid chromatogram) of the free type beta-cryptoxanthin composition concerning an embodiment of the invention. It is a graph which shows the 1H nuclear magnetic resonance spectrum of the free type beta-cryptoxanthin composition concerning an embodiment of the invention. (A) is a graph of a standard sample. (B) is a graph of the free β-cryptoxanthin composition of the present embodiment. It is a figure which shows the yield of the (beta) -cryptoxanthin concentrated composition manufactured from the Citrus unshiu peel which concerns on embodiment of this invention, a content, content rate, and a recovery rate. It is a figure which shows the yield, content, and content rate of the carotenoid concentration composition in the oyster peel which concerns on embodiment of this invention. It is a figure which shows the yield, content, and content rate of the free type | mold carotenoid composition obtained by hydrolysis of the high concentration carotenoid composition derived from the oyster peel which concerns on embodiment of this invention.

<Embodiment>
[Method of extracting carotenoid composition]
As described above, the inventors of the present invention have conducted intensive studies and experiments in order to develop a production method for obtaining a β-cryptoxanthin composition having a concentration that can be used for pharmaceuticals, quasi drugs, supplements, or food additives. Went.
At this time, the inventors used foods such as vegetables and fruits containing carotenoids. For example, a process for processing a fruit containing β-cryptoxanthin as a carotenoid, that is, mandarin oranges, for example, Citrus unshiu Marc., Citrus tangerina Hort. By-products such as pericarp and pulp produced in fruit juice production processes such as reticulata blanco) and fruit processing processes such as oysters were used as raw materials.
And the inventors can remove components other than the mixed carotenoid as much as possible without using means such as chromatographic methods by optimizing the combination of the extraction solvent and the extraction temperature. We have found a method for obtaining a carotenoid composition at a concentration that can be used for pharmaceuticals, quasi drugs, supplements, or food additives in a short time.
Furthermore, the inventors have also found a method for producing a high-concentration carotenoid composition from this carotenoid composition. In addition, based on this high-concentration carotenoid composition, free carotenoids that contain almost no components other than carotenoids can be easily obtained without using advanced chromatographic methods such as high-performance liquid chromatography. I found a way to do it.
Accordingly, the inventors have obtained a carotenoid composition containing β-cryptoxanthin or other carotenoid according to an embodiment of the present invention at a concentration that can be used for pharmaceuticals, quasi drugs, supplements, or food additives. A manufacturing method for manufacturing at low cost has been completed.
Hereinafter, a method for extracting a β-cryptoxanthin composition mainly using the method for extracting a carotenoid composition of the present embodiment will be described in detail with reference to the process chart (flow chart) of FIG. 1.

(Pretreatment of raw material for extraction)
First, in the process of step S101, the raw material of the extraction raw material is pretreated.
Specifically, the raw material used in the method for extracting β-cryptoxanthin according to the embodiment of the present invention is not limited to plants and food itself, and may be a by-product such as a peel generated during processing of the food material. It does not depend on the composition of the carotenoids contained.
However, it is particularly preferable to use mandarin oranges containing β-cryptoxanthin as the main carotenoid, for example, peeled skin or pulp generated during squeezing of Citrus unshiu, because it contributes to effective use of resources.
In addition, fruit peels produced during the production of dried persimmons of oysters containing lutein, zeaxanthin, β-cryptoxanthin, and β-carotene are desirable from the viewpoint of resource utilization.
In order to remove moisture, these extraction materials are dried by a known freeze-drying apparatus, reduced-pressure drying, drum-type food drying apparatus, powder mist-type drying apparatus, or the like. The water content at this time is preferably 5% or less from the viewpoint of extraction efficiency.

In addition, as peeled skin, as usual, slurries by enzymatic treatment, dried products collected by centrifugation, or dried by warm air or freezing, etc. after blanching according to the conventional method of herbal medicine preparation It may be used.
More specifically, the pericarp is, for example, roughly crushed pericarp residue generated in the processing step of squeezed juice or canned fruit, then boiled (90 ° C.), and squeezed or centrifuged. Except for water, a dry product having a water content of 5% or less obtained by hot air drying, freeze drying or other drying methods can be used as a powder.
In the case of oysters, a portion derived from the pericarp, such as peeled pericarp generated in the processing step of dried persimmons, dried using warm air, freeze drying or other drying methods may be used.

As the pulp, finisher pulp during sieving, centrifugal pulp, and diatomaceous earth adsorbing pulp for filtration during fruit juice clarification treatment can be used.
More specifically, as this pulp, after removing moisture from the pulp that is a by-product generated by sieving or centrifuging in the juice extraction process, for example, by the centrifugal process, etc. A dry product having a water content of 5% or less obtained by hot air drying, freeze drying or other drying methods can be used as a powder.
Water content obtained by recovering diatomaceous earth adsorbing insoluble matter generated in the filtration process during the production of clear juice and removing water by centrifugation, etc., followed by hot air drying, freeze drying, or other drying methods 5% or less of a dried product can be used as a powder.
In these pulps, washing and centrifuging are repeated, and the precipitate is freeze-dried or heat-dried after the Brix ° is 2 (degrees) or less, which represents the solid content concentration in the solution of the washing solution. Can do. Brix ° (degree) is a scale (scale) name of the refractive index converted to a weight percentage concentration (W / W%) of the sucrose solution at 20 ° C.
These extraction materials can be used as raw materials for carotenoid extraction.

(Extraction of fat-soluble substances from raw materials)
Next, in step S102, a fat-soluble substance containing carotenoid is extracted from the raw material.
Specifically, with reference to FIG. 2, in the present embodiment, carotenoid extraction is performed simultaneously with the extraction of the fat-soluble substance in the raw material.
FIG. 2 is an experimental example using Satsuma mandarin pulp. Hereinafter, in the graphs of FIGS. 2 to 11, the “○” circles in the graph indicate the soluble part (fraction of soluble components) that can be extracted with respect to the solvent, and the “□” squares indicate the insoluble part (fraction of insoluble components). Draw as precipitation).
The vertical axis | shaft of FIG. 2 shows content of the various fat-soluble substance contained in the extract distributed to the soluble part, or the extract distributed to the insoluble part as distribution amount (%). That is, the distribution amount indicates the weight% of the fat-soluble substance contained in the extracted extract or extract. This fat-soluble substance contains β-cryptoxanthin.
The horizontal axis of FIG. 2 shows the content (%) of hexane in an extract composed of pure hexane and 99.5% ethanol (hereinafter simply referred to as ethanol). The left end is hexane: ethanol = 100%: 0%, and the right end is hexane: ethanol = 0%: 100%.

According to FIG. 2, when the mixing ratio of hexane and ethanol is changed and extraction is performed until the carotenoid is hardly colored in the sample, the carotenoid extracted in the soluble part is the largest in extraction with 100% hexane. It turns out that it is. Conversely, when the ethanol content is increased to 40% or more, carotenoids are included in the insoluble portion. For this reason, extraction of a fat-soluble substance with hexane 100% is the most desirable extraction condition.
At the time of this extraction, there is almost no difference in the extraction amount depending on the extraction temperature between −20 ° C. and 18 ° C.
Therefore, extraction of this fat-soluble substance has no problem with extraction at room temperature. Further, the hexane extract is distilled off under reduced pressure to obtain an oily hexane extract (concentrate). The hexane at this time can be recovered and used again for the extraction operation.
The extraction solvent may be extracted with hexane after adding an appropriate amount of ethanol depending on the state of the extraction raw material.

  In addition, about extraction of the fat-soluble substance from peeled skin, since essential oil is contained in the skin as will be described later, ethanol 10% to 20%, water 80 to 90%, about 5 times the weight of hexane extract. In addition, it is preferable to obtain a concentrate by distillation under reduced pressure while heating at 50 ° C to 60 ° C.

(Production of low-concentration carotenoid composition)
Next, in the process of step S103, a low concentration carotenoid composition is manufactured.
Specifically, 2 to 3 times the amount of redissolved solution is added to the oily hexane extract (concentrate) extracted in the above-described extraction step of the fat-soluble substance from the raw material, and the resulting precipitate is centrifuged. (4500 rpm, 4 min, room temperature, etc.) to recover the soluble part. As this redissolved solution, it is preferable to use 100% hexane.
Below, with reference to FIGS. 3-7, the manufacturing method of a low concentration carotenoid composition is demonstrated in detail.

First, with reference to FIG. 3, the result of having examined the mixing ratio of hexane and ethanol of the redissolved solution will be described.
FIG. 3 is a diagram in which the distribution ratios of the soluble part and the insoluble part of the fat-soluble substance to the respective fractions were examined using the redissolved solution. In FIG. 3, as in FIG. 2, the vertical axis represents the distribution amount (%), and the horizontal axis represents the hexane content (%) of the redissolved solution.
As shown in this result, it was found that when the ethanol mixing ratio was 0 to 40%, there was no significant change in the value of the distribution rate. That is, in the concentration of hexane: ethanol = 100%: 0% to hexane: ethanol = 60%: 40%, there was no significant change in the distribution of the fat-soluble substance between the soluble part and the insoluble part.

Next, with reference to FIG. 4, the distribution of β-cryptoxanthin to the soluble part and the insoluble part will be described.
Specifically, FIG. 4 shows the measurement results for the distribution ratio of β-cryptoxanthin to the soluble and insoluble parts in each redissolved solution distributed by changing the mixing ratio of hexane and ethanol in FIG. Is shown. The vertical axis | shaft of FIG. 4 has shown the ratio (%) of (beta) -cryptoxanthin contained in a soluble part and an insoluble part. That is, the ratio of soluble part (%) + the ratio of insoluble part (%) = 100 (%). Moreover, the horizontal axis of FIG. 4 shows the content (%) of 99.5% ethanol in the redissolved solution. That is, hexane: ethanol = 100%: 0% at the left end of FIG. 4, and hexane: ethanol = 0%: 100% at the right end of FIG.
In FIG. 4, no significant change is observed between the ethanol contents of 0 to 50%, and most of them are distributed in the soluble part.
The distribution is reversed around the ethanol concentration of 90%, and 80% of β-cryptoxanthin is distributed in the insoluble portion at 100% ethanol.
For this reason, 50%-100% hexane is suitable as a re-dissolution liquid.

Next, with reference to FIG. 5, the relationship between the cooling temperature at the time of remelting and the weight of the soluble part / insoluble part will be described. Here, redissolving in a 100% hexane solution was performed. In FIG. 5, the vertical axis indicates the weight ratio (%) between the soluble part and the insoluble part. That is, soluble part + insoluble part = 100 (%). Further, the horizontal axis of FIG. 5 indicates the cooling temperature.
As shown in FIG. 5, the cooling temperature at the time of hexane fractionation and the ratio between the soluble part and the insoluble part change linearly. That is, the lower the temperature, the more precipitates.

Next, with reference to FIG. 6, the relationship of the content of β-cryptoxanthin in the soluble part / insoluble part obtained at the time of re-dissolution in FIG. 5 will be described. In FIG. 6, the vertical axis indicates the content (mg / 100 g) of β-cryptoxanthin per 100 g of the soluble part and the insoluble part, and the horizontal axis indicates the same cooling temperature as in FIG.
As shown in FIG. 6, the relation between the content of β-cryptoxanthin per unit weight in the soluble part and the insoluble part and the cooling temperature is almost dissolved in the soluble part between −10 and −30 ° C. ing.

Next, with reference to FIG. 7, the content rate of (beta) -cryptoxanthin in the soluble part / insoluble part obtained at the time of re-dissolution of FIG. 5 is demonstrated. In FIG. 7, the vertical axis represents the content (%) of β-cryptoxanthin per weight of the soluble part and the insoluble part. Moreover, the horizontal axis of FIG. 7 shows the same cooling temperature as FIG.
As shown in FIG. 7, the carotenoid content in the soluble part and the insoluble part becomes an extreme value in the vicinity of −30 ° C.
In addition, in the manufacturing process of the low concentration carotenoid composition of this embodiment, it aims at removing components other than carotenoid. That is, it is not intended to precipitate carotene by concentration as disclosed in the carotene concentration method of Patent Document 8.

Thus, the first stage extraction in the production of the low-concentration carotenoid composition in step S103 can be achieved by using hexane and obtaining a soluble part obtained by cooling at -20 ° C to -30 ° C.
The low-concentration carotenoid composition obtained here is a dark red substance that is oily at room temperature and when frozen.

(Production of medium concentration carotenoid composition)
Next, in the process of step S104, a medium concentration carotenoid composition is manufactured.
In this step, 29.5 to 4 times the amount of 99.5% ethanol in terms of weight is added to the low-concentration carotenoid composition obtained in the above step, cooled, and the resulting insoluble portion is centrifuged (4500 rpm, 4 min). Room temperature) and collected. Thereby, a medium concentration carotenoid composition is obtained. This medium-concentration carotenoid composition has a concentration that can be sufficiently used as a pharmaceutical, a health supplement, a quasi drug, and a food additive.
Below, with reference to FIGS. 8-10, the manufacturing method of a low concentration carotenoid composition is demonstrated in detail.

First, with reference to FIG. 8, the cooling temperature at the time of redissolving ethanol and the weight ratio (%) of the soluble part / insoluble part will be described.
In FIG. 8, the vertical axis represents the weight ratio (%) between the soluble part and the insoluble part, and the horizontal axis represents the cooling temperature (° C.), as in FIG.
As shown in FIG. 8, when redissolving in 99.5% ethanol, the relationship between the cooling temperature at the time of fractionation and the weight of the soluble and unnecessary parts changes linearly.

Next, with reference to FIG. 9, the β-cryptoxanthin content in the soluble part and the insoluble part at the time of ethanol redissolution will be described.
In FIG. 9, as in FIG. 6, the vertical axis represents the content (mg / 100 g) of β-cryptoxanthin per 100 g of the soluble part and the insoluble part, and the horizontal axis represents the cooling temperature.
As shown in FIG. 9, the amount of carotenoid distributed to the soluble part and the insoluble part has an inflection point from -20 to -30 ° C.

Next, with reference to FIG. 10, the β-cryptoxanthin content (%) in the soluble part and the insoluble part at the time of ethanol redissolution will be described.
In FIG. 10, as in FIG. 7, the vertical axis represents the content (%) of β-cryptoxanthin per weight of the soluble part and the insoluble part, and the horizontal axis represents the cooling temperature.
As shown in FIG. 10, the carotenoid content in the insoluble portion has an extreme value at −20 ° C. to −30 ° C.

Thus, it has been found that the second stage of extraction by the production process of the medium concentration carotenoid composition in Step S104 can be achieved by fractionating insoluble components obtained by cooling to −20 ° C. to −30 ° C. using ethanol. .
The product obtained here is an oily dark red substance at room temperature and when frozen.

(Manufacture of high-concentration carotenoid composition)
Next, in the process of step S105, a high-concentration carotenoid composition is manufactured.
In this step, 2 to 3 times the amount of hexane / ethanol mixture is added to the medium concentration carotenoid composition obtained in the above step, cooled to −20 to −30 ° C., and the resulting insoluble part is centrifuged. Separate (4500 rpm, 2 min, room temperature) and collect.

Here, with reference to FIG. 11, the hexane / ethanol mixing ratio and the β-cryptoxanthin content (%) in the soluble and insoluble parts will be described.
The vertical axis | shaft of FIG. 11 shows the content rate (%) of (beta) -cryptoxanthin in each weight in a soluble part and an insoluble part.
The horizontal axis of FIG. 11 shows the ethanol content (%) of an extract composed of pure hexane and 99.5% ethanol. The left end is hexane: ethanol = 100%: 0%, and the right end is hexane: ethanol = 0%: 100%.
As shown in FIG. 11, the relationship between the composition of the hexane / ethanol mixture and the carotenoid content in the insoluble part shows that hexane: ethanol = 40%: 60% has a maximum. It is effective to draw.
The product obtained in the production process of the high-concentration carotenoid composition is an oil at room temperature and is a dark red substance that solidifies upon freezing.
If necessary, the insoluble matter after centrifugation can be redissolved in a hexane: ethanol = 40%: 60% solution and fractionated again.

(Production of high purity free carotenoid composition)
Next, in the process of step S106, a high-purity free carotenoid composition is produced.
The above-described high-concentration carotenoid composition is a high-concentration ester type carotenoid composition. That is, carotenoids exist in a state in which they are ester-bonded with fatty acids.
By hydrolyzing this ester bond with potassium hydroxide in an organic solvent using a known method, only a pure carotenoid can be extracted to produce a high purity free carotenoid composition.

Here, when a free carotenoid is produced from a carotenoid composition by alkaline hydrolysis, it is reasonable to use a medium or high concentration carotenoid composition.
Specifically, after adding t-butyl methyl ether to a high-concentration carotenoid composition and dissolving it, a third amount of 10% potassium hydroxide / methanol solution was added and mixed, followed by nitrogen substitution and overnight at room temperature. put.
T-Butyl methyl ether is added to the reaction product, water is added, and the mixture is stirred and then divided into an organic phase and an aqueous phase by centrifugation. The aqueous phase is removed, water is added again, and the aqueous phase is removed again.
This operation is repeated several times until the aqueous phase is neutral, and then the organic phase is concentrated under reduced pressure to obtain a dark red residue.
About 10 mL of n-hexane is added to 1 g of the residue, dispersed in an ultrasonic bath, and the precipitate is recovered by filtration or centrifugation.
By filtering the fine insoluble matter produced at this time, a purple-colored substance having a metallic luster can be obtained.
If necessary, add about 10 mL of ethanol to the recovered material, heat to about 60 ° C., disperse in an ultrasonic bath, and filter the fine insoluble matter produced during cooling to give a purple-colored metallic luster. A substance can be obtained.
The obtained substance becomes a high-concentration free carotenoid composition containing almost no components other than carotenoids.
Thus, each step of the carotenoid composition extraction method is completed.

  The extracted carotenoid composition is, for example, a high-concentration free β-cryptoxanthin composition in the case of Citrus unshiu, so that it is efficient as various supplements such as pharmaceuticals, health supplements, quasi drugs, and food additives. Can be used well.

(Method for producing free carotenoids from peel extract of Citrus unshiu)
In the case of Citrus unshiu peel extract, when a high-concentration free carotenoid composition is extracted by the above-described production method, the yield is about 2%.
Although free cryptoxanthin obtained by hydrolysis from this high-concentration carotenoid composition is crystallized only by the operation of the solvent, the yield is low due to contamination of lipids.
On the other hand, it can be easily crystallized by passing through a silica gel column once.

The case where free carotenoids are produced from Citrus unshiu peel will be described more specifically below.
The high-concentration carotenoid composition derived from the pericarp contains a large amount of fat including fats and oils (about 35%, calculated from the integral intensity ratio of glycerol methylene proton and ethanol methylene proton added as an internal standard substance by NMR method). include.
Since these lipids are liquid at room temperature, in addition to hexane and ethanol, the three components of lipids are solvents. This composition is completely mixed depending on the mixing ratio of hexane and ethanol, or has two phases. However, carotenoids are dispersed in both phases due to the influence of lipids and cannot be obtained as an insoluble matter.
For this reason, when the peel is used, it is difficult to increase the concentration exceeding 10% only by optimizing the composition of hexane and ethanol, as in the case of using pulp, and the concentration remains at about 2% on average. In other words, when free carotenoids are obtained from a composition (concentrate) containing a large amount of such lipids, high purity free carotenoids can be obtained as crystals by a combination of solvents, but they are mixed with lipids even after alkaline hydrolysis. Therefore, it is difficult to obtain a high yield as in the case where the above-mentioned pulp is derived.
Therefore, separation by simple column chromatography using silica gel is performed. In this separation, the solvent used for elution may be a combination of hexane and acetone, which is not a non-medical deleterious substance and has not been suspected of causing carcinogenicity. That is, it is not necessary to use a sophisticated chromatographic method like a high-performance liquid chromatograph, and further, it is not necessary to use a solvent that is an external pharmaceutical product such as acetonitrile.
More specifically, fractionation is performed once using the dark red band as an index, the fraction is concentrated to dryness with an evaporator, ethanol is added, and the mixture is heated and dissolved, and then left in a cool dark place to facilitate β- Cryptoxanthine crystals can be obtained.

In addition, about the crystal mother liquid which occurs when it precipitates only by the operation of the solvent in the high purity free β-cryptoxanthin production process derived from pulp, recrystallization is possible because the concentration of free β-cryptoxanthin is low after hydrolysis. If difficult, free β-cryptoxanthin can also be concentrated using separation by the simple silica gel column chromatography method described above.
As a result, the remaining free β-cryptoxanthin can be crystallized again to increase the recovery rate.

With the configuration described above, the following effects can be obtained.
Conventionally, in order to obtain a high-concentration β-cryptoxanthin composition, there has been a problem that an expensive method such as a high performance liquid chromatographic method must be used.
On the other hand, the carotenoid composition extraction method according to the embodiment of the present invention does not use a costly technique such as a high performance liquid chromatographic method, and does not provide a high concentration β- A cryptoxanthin composition can be produced.
Thereby, (beta) cryptoxanthin can be utilized cheaply for a pharmaceutical, a quasi-drug, a supplement, or food additive.

In addition, in the carotenoid composition extraction method according to the embodiment of the present invention, the raw material includes xanthophylls such as lutein, zeaxanthin, and violaxanthin, and hydrocarbon carotenoids such as β-carotene other than β-cryptoxanthin. In some cases, a concentrate corresponding to the composition can be obtained.
Thereby, the carotenoid composition of the density | concentration which can be used for a pharmaceutical, a quasi-drug, a supplement, or a food additive can be obtained, for example using an oyster peel.

In addition, the carotenoid composition extraction method according to the embodiment of the present invention uses two types of solvents, hexane and ethanol, which are recognized as food additives, and devise the composition, the extraction order, and the extraction temperature. It can be produced by extracting a composition containing a high concentration of type β-cryptoxanthin.
Thereby, a carotenoid composition for safe pharmaceuticals, quasi drugs, supplements, or food additives can be obtained without using a carcinogenic organic solvent or the like.

In addition, the carotenoid composition extraction method according to the embodiment of the present invention can be easily expanded on an industrial scale.
First, the above-mentioned raw material pulp is dehydrated by squeezing or centrifuging the squeezed residue consisting of the pericarp and fungus generated by in-line squeezing of mandarin oranges, sieving and centrifuging It is a by-product generated by doing.
The pulp can be removed together with sugar and water-soluble components by washing with water and centrifuging with a general industrial press or household dehydrator.
Further, it can be sufficiently dried by an industrial warm air dryer or the like without using a freeze dryer. Similarly, the pulverizer is a general device and has no problem.

In the extraction of fat-soluble substances from raw materials, the pulverized raw material is put into an extraction tube such as stainless steel, hexane is dropped from the top to collect the eluate, and the recovered hexane is concentrated by an evaporator or the like. It is preferred to circulate again for extraction. Thereby, extraction operation can be performed without waste with a small amount of hexane.
Moreover, volatilization of hexane can be suppressed by putting the concentrate into a centrifuge container, re-dissolving in hexane, centrifuging after cooling, and collecting the supernatant.
Concentration of the supernatant is easy, and redissolving in ethanol and recovery of insoluble matter after cooling can be performed in the same manner as in the above hexane treatment. Furthermore, the insoluble portion can be recovered by cooling with a mixed solution of hexane and ethanol without changing the container in the same manner as the ethanol treatment.
Also, the production of high purity free β-cryptoxanthin can be carried out as it is in the above container. It is also easy to collect the hydrolysis product by mixing the sodium chloride solution in the container and performing centrifugation after mixing, and by inserting the nozzle of the solution suction device to collect only the necessary phase.
Separation by a simple column chromatographic method can be easily expanded to an industrial scale because a target product can be obtained visually by using a glass column without using a detector or the like.

Conventionally, it has been known that carotenoids are also contained in by-products such as pericarp generated in the processing process of fruits, but it has not always been effectively utilized and utilized.
On the other hand, the carotenoid composition extraction method according to the embodiment of the present invention effectively utilizes the peel produced during the production of Citrus unshiu juice, various forms of pulp, pharmaceuticals, health supplements, quasi drugs, A high-concentration β-cryptoxanthin ester type composition that can be efficiently used for food additives can be obtained.
Specifically, a β-cryptoxanthin composition having a high concentration of 4% (on the average, about 2%) from the peel and about 14% from the pulp can be obtained. This is an amount equivalent to 67% of the extracted raw material in terms of free conversion as a recovery rate from the raw material of β-cryptoxanthin, and using raw materials previously discarded such as leather and pulp, Can be used effectively.
Moreover, since this high concentration β-cryptoxanthin ester type composition can be sold, it becomes possible to reduce the squeezing cost on the balance.

Further, by hydrolyzing a high concentration β-cryptoxanthin ester type composition, free β-cryptoxanthin can be obtained as crystals without performing separation by a chromatographic method.
In the case of a high-concentration β-cryptoxanthin ester-type composition derived from the peel, free β-cryptoxanthin can be obtained as crystals only by performing a single chromatographic separation using silica gel as a carrier. Can do. At this time, it is not necessary to use an organic solvent such as acetonitrile which is a non-medical deleterious substance.
Moreover, the xanthophyll composition containing a high concentration of β-cryptoxanthin can be obtained by similarly treating carotenoids contained in oyster peels generated by processing such as dried persimmons.
Thereby, the free carotenoid composition which can be utilized as a pharmaceutical, a health supplement, a quasi-drug, and a food additive at low cost can be obtained.

  Next, the method for producing the carotenoid composition according to the embodiment of the present invention will be described more specifically with reference to the following examples. However, this embodiment is only an example, and the present invention is not limited to this.

[Experiment and detection method]
(Preparation of carotenoid analysis sample)
First, preparation of the carotenoid analysis sample will be described. As a pretreatment of the raw material for extraction, 10 g of fresh fruit is precisely measured, and 50 mL of hexane: acetone: ethanol = 50%: 25%: 25% extraction solvent is added and mixed vigorously with a homogenizer under ice cooling (30 Seconds).
In the case of a dried fruit sample, 1 g is accurately weighed and 10 mL of water is added, and then the same operation is performed.
In the following, Wako Pure Chemical Industries (special grade) was used for hexane, ethanol, and acetone.

Next, the mixture was transferred to a polypropylene centrifuge tube (50 mL, 2 tubes), and the insoluble content (insoluble portion) was removed by centrifugation at 4500 rpm for 10 minutes at 25 ° C. (LC-220, manufactured by Tommy Seiko Co., Ltd.). Sink.
And the upper layer (hexane layer) which is a soluble part is collect | recovered, and hexane (5 mL, 2 times) is then added to the slightly remaining upper layer, and it collect | recovers. The obtained upper layer is combined with an eggplant-shaped flask (100 mL), and concentrated under reduced pressure at 30 ° C. with a rotary evaporator (Tokyo Rika Kikai, N-1000S type).
Next, the concentrated upper layer is returned to atmospheric pressure with nitrogen, and the concentrate is dissolved in 0.1% butylhydroxytoluene-containing t-butyl methyl ether (manufactured by Wako Pure Chemical Industries, Ltd., for HPLC), and polypropylene Transfer to a settling tube (15 mL). Then, t-butyl methyl ether (Wako Pure Chemical Industries, HPLC) is added to make 3 mL.
Next, 10% methanolic potassium hydroxide (2 mL) is added to the obtained t-butyl methyl ether solution, the inside of the centrifuge tube is purged with nitrogen, and the mixture is allowed to stand overnight at room temperature under light shielding for hydrolysis. The potassium hydroxide solution used was a special grade manufactured by Wako Pure Chemical Industries.
The hydrolyzed reaction solution was transferred to a separating funnel (100 mL) containing water (50 mL), and the hydrolyzed centrifuge tube was added with 0.1% butylhydroxytoluene-containing t-butyl methyl ether (3 mL, 2 times). Wash and match with a separatory funnel.
Next, the separatory funnel is allowed to stand after shaking, the separated aqueous layer is removed, the organic layer is washed twice with water (10 mL), the organic layer is recovered, and the evaporator solvent is distilled off under reduced pressure.
The residue after removal of the solvent was dissolved in 0.1% butylhydroxytoluene-containing t-butyl methyl ether: ethanol mixture = 1: 1 to 2.0 mL, and this was filtered through a PTFE (0.2 μm) filter. Transfer to analytical vial. The space in the vial is replaced with nitrogen gas. (The method described above is based on the method of Lee Castle (2001) (J. Agric. Food Chem. 49: 877-882) with some modifications).

Carotenoid analysis in the fractionation solution was carried out using 15 mL polypropylene with a predetermined volume of each fraction, measuring a predetermined amount (10 to 20 μL) with a microsyringe, and containing 2 mL of 0.1% butylhydroxytoluene-containing t-butyl methyl ether. In addition to the centrifuge tube, 1 mL of a 10% potassium hydroxide methanol solution was added and the atmosphere was purged with nitrogen.
Then, 8 mL of water was added and mixed and centrifuged (4500 rpm, 1 minute, room temperature), water was added to make the lower layer 10 mL, t-butyl methyl ether containing 0.1% butylhydroxytoluene was added, and the upper phase was 2 mL. did. Then, a predetermined amount (100 μL to 1 mL) is taken from the upper phase with a microsyringe according to the concentration, transferred to a volumetric flask, ethanol is added to 2 mL, and this is analyzed by HPLC (High performance liquid chromatography, high performance liquid chromatograph). A sample was prepared.

(Carotenoid HPLC analysis method)
Carotenoid analysis by the HPLC method was performed by modifying the method of Sander et al. (1994) (Anal. Chem. 66: 1667-1674). For the HPLC apparatus, HP1100 manufactured by Agilent was used. The measurement conditions are as follows.
Column, Carotenoid S-3μ (3.0 mm id × 150 mm, Waters) manufactured by YMC. Column temperature, 30 ° C.
Solvent system, liquid A: methanol: acetonitrile: water = 7: 1: 2, liquid B: t-butyl methyl ether: methanol = 9: 1. The methanol, acetonitrile, and t-butyl methyl ether were used for Wako Pure Chemical Industries, HPLC. Moreover, the standard product by DHI Water & Environment used the carotenoid standard product for HPLC analysis.
Concentration gradient program, solution B concentration is maintained at 22.5% for 0.5 minutes after the start of analysis and is linearly increased to 65% in 44.5 min.
Flow rate, 1.0 mL / min. Sample injection volume, 2 μl. Detection wavelength: 455 nm. The content was quantified by the absolute calibration curve method.

(Analysis with a nuclear magnetic resonance apparatus)
Analysis was performed using an Avance DPX400 manufactured by Bruker as a nuclear magnetic resonance apparatus.
For the nuclear magnetic resonance spectrum, each fraction solution was concentrated, about 10 mg was weighed, dissolved in deuterated chloroform, and tetramethylsilane vapor was blown into the solution to make a δ value standard.

[Production of highly concentrated carotenoid composition using Citrus unshiu]
Example 1
An example in which a highly concentrated carotenoid composition containing β-cryptoxanthin is produced using the pericarp of Citrus unshiu Marc as a raw material using the treatment according to the above-described embodiment will be described.
First, the peel of Citrus unshiu (the Citrus unshiu cultivated by Orchard Laboratories) was roughly pulverized with a food cutter (Food Machine Instrument, R3-1500), and then poured into boiling water to stop heating, sometimes for 15 minutes. Water was poured while stirring.
Then, the skin was collected with a nylon mesh, moisture was removed by pressing or dehydrating machine, and dried for about 12 hours with a warm air dryer set at 50 ° C. The moisture content of this dried product was 5% or less when calculated from the weight loss after vacuum drying under vacuum. The dried product of the peeled skin was made into a fine powder with a pulverizer (Waring Blender 7011HS, manufactured by Waring).
Then, it is placed in a glass column tube with a diameter of 10 cm and a length of 80 cm with a glass filter, extracted with 3 L of hexane, and about 5 times as much ethanol: water = 2: 8 is added to the concentrate and heated at 60 ° C. The crude extract as an oily residue was obtained by distillation under reduced pressure using an evaporator. This operation was performed to distill away essential oil components such as limonene together with water.
After adding approximately 3 times the amount of hexane to this crude extract and cooling at −30 ° C. for 1 day, it was centrifuged at 4500 rpm for 3 minutes at room temperature, and the soluble part of the upper layer was recovered and concentrated under reduced pressure. Oil was obtained.
About 3 times the amount of ethanol was added to this oily substance, dispersed in an ultrasonic bath (USC-1.5D manufactured by Iwaki Co., Ltd.), and cooled at -30 ° C for 1 day. Thereafter, the mixture was centrifuged at 4500 rpm for 3 minutes at room temperature, and the lower insoluble part was collected and concentrated under reduced pressure with an evaporator to obtain an oily substance.
This oily substance was a highly concentrated carotenoid composition containing at least 2.0% by weight of β-cryptoxanthin (in terms of free β-cryptoxanthin).

(Example 2)
Next, referring to FIG. 12, an example of producing a highly concentrated carotenoid composition containing β-cryptoxanthin using centrifugal pulp (manufactured by Nippon Fruit Industry) at the time of sieving (sieving) Citrus unshiu juice as a raw material. Will be described. The production process was carried out in the same manner as in Example 1, and finally an oily substance containing β-cryptoxanthin was obtained.
In the example of FIG. 12, for each step, the yield (g), which is the obtained amount of each composition, the content (g) of β-cryptoxanthin, the content /% (yield / content), and the oil recovery (%) Is shown. Each value was measured by preparing a sample using the above-described method for preparing a carotenoid analysis sample.

Referring to FIG. 12, in Example 2, in the obtained low-concentration carotenoid composition, the β-cryptoxanthin content was about 3.6% as a free form, and the recovery rate from the raw material was 96% or more. Here, the recovery rate indicates how much β-cryptoxanthin contained in the raw material remains and is contained in the composition of each step.
Further, in the medium concentration carotenoid composition, the β-cryptoxanthin content was about 4.9% as a free form, and the recovery rate from the raw material was 92%. This was the best recovery when high concentrations were not required.
In the high-concentration carotenoid composition, the β-cryptoxanthin content was about 13% as a free form, and the recovery rate from the raw material was 75% or more.
Thus, the carotenoid composition which contains (beta) -cryptoxanthin by the density | concentration higher than before can be manufactured efficiently by using the centrifugal pulp of Satsuma mandarin.

(Example 3)
Next, with reference to FIG. 13, the example which manufactured the highly concentrated carotenoid composition containing (beta) -cryptoxanthin using the filtration process diatomaceous earth generated at the time of manufacture of the clear juice (manufactured by Nippon Fruit Industry) of Citrus unshiu Will be described.
The production process was the same as in Example 1 above, and finally an oily substance containing β-cryptoxanthin was obtained. In the example of FIG. 13 as well, each value is shown for each process, as in FIG.

Referring to FIG. 13, in Example 3, the low-concentration carotenoid composition had a β-cryptoxanthin content (as a free form) of about 2.1% and a recovery rate from the raw material of 91% or more.
The medium-concentration carotenoid composition had a β-cryptoxanthin content (as a free form) of about 6.5% and a recovery rate of about 80%. For this reason, as in Example 2, when the high concentration was not required, the recovery rate was the best.
The high-concentration carotenoid composition had a β-cryptoxanthin content (as a free form) of about 14% and a recovery rate from the raw material of 66% or more.
In this way, even if you use filtration diatomaceous earth of Satsuma mandarin. A carotenoid composition containing β-cryptoxanthin can be produced with high efficiency.

[Production result of high purity free β-cryptoxanthin derived from Citrus unshiu pulp]
Next, with reference to FIGS. 14-15, the result of manufacture of the high purity free type | formula-cryptoxanthin obtained from the unshu citrus pulp (Nippon Fruit Industry) of the above-mentioned Example 2 is demonstrated.
1 g of high-concentration ester type carotenoid composition obtained from Satsuma mandarin pulp of Example 2 was dissolved in 30 mL of t-methylbutyl ether (manufactured by Wako Pure Chemical Industries, Ltd., for HPLC), and then 20 mL of 10% potassium hydroxide methanol. The solution was added, mixed after nitrogen substitution, and allowed to stand overnight in the dark at room temperature for hydrolysis.
Transfer the hydrolyzed reaction liquid to a separatory funnel, add t-butyl methyl ether as appropriate, add water and stir, wash the organic phase with water, place it in a 250 mL centrifuge bottle, centrifuge at 4500 rpm for 4 minutes, and remove the aqueous phase. Separated and removed. This operation was repeated several times, and the organic phase was recovered when the water washes became neutral.
Thereafter, the recovered organic phase was concentrated with a rotary evaporator, hexane was added, and the mixture was dispersed by sonication (using USC-1.5D manufactured by Iwaki) and left standing. As a result, dark purple crystals having a metallic luster were deposited.
This dark purple crystal was centrifuged at 4500 rpm for 4 minutes to remove the hexane-soluble part, ethanol was added to the precipitate as appropriate and dispersed by ultrasonic treatment, and ethanol treatment was performed to collect the dispersed crystal by filtration.
Thereafter, the crystals collected after the ethanol treatment were analyzed by a high performance liquid chromatography method (FIG. 14) and a nuclear magnetic resonance apparatus (FIG. 15).

FIG. 14 shows the HPLC results of the free β-cryptoxanthin product. In the results shown in FIG. 14, an almost single large peak of β-cryptoxanthin was detected.
Further, FIG. 15 shows the result of analysis by 1H nuclear magnetic resonance spectrum of a pure β-cryptoxanthin pure product. Here, FIG. 15 (a), which is the upper graph, shows the analysis result of a control pure β-cryptoxanthin standard sample (manufactured by Shikoku Yasushi Pharmaceutical Co., Ltd., the peak near δ3.5 is derived from the remaining impurities). It is. FIG. 15B, which is a lower graph, shows the analysis result of the high-purity free β-cryptoxanthin obtained in this example. FIG. 15B shows that substantially the same size and spectrum peak can be detected, and that substantially pure β-cryptoxanthin can be obtained.
From the above results, almost pure (96%) free β-cryptoxanthin (91.8 mg) could be identified.
Thus, free β-cryptoxanthin can be obtained from the raw material high-concentration ester β-cryptoxanthin composition at a recovery rate of about 65%.
The ethanol treatment described above can be omitted depending on circumstances. When it is desired to further increase the degree of purification, recrystallization can be performed by heating to about 60 ° C. during ethanol treatment and dissolving.

[Production result of high-purity free β-cryptoxanthin without using t-methylbutyl ether and methanol during hydrolysis]
In the above-mentioned Examples, at the time of hydrolysis, extraction of a high purity free-type carotenoid composition was performed using t-methylbutyl ether and methanol.
On the other hand, it was also possible to obtain high purity free β-cryptoxanthin without using t-methylbutyl ether and methanol.
For example, a high-concentration ester-type carotenoid composition (10 g) is dissolved in hexane (100 mL), 10% potassium hydroxide ethanol solution (20 mL) is added and mixed, purged with nitrogen, and left in the dark at room temperature for 4 hours. Red needle crystals were precipitated.
When this was collected by filtration and subjected to HPLC analysis (not shown), almost pure (96%) free β-cryptoxanthin (1.3 g) could be obtained.
By comprising in this way, it becomes possible to obtain high purity free beta-cryptoxanthin at low cost only by the manufacturing process using hexane / ethanol extraction.

[Production result of high purity free β-cryptoxanthin derived from Citrus unshiu peel]
Example 4
After dissolving 6.62 g of high-concentration ester type carotenoid composition obtained from Satsuma mandarin peel of Example 1 in hexane (100 mL), 10% potassium hydroxide ethanol solution prepared at the time of use (20 mL) was added and mixed. After purging with nitrogen, the mixture was allowed to stand overnight in the dark at room temperature for hydrolysis.
After insoluble matter in the reaction solution is centrifuged (4500 rpm, 4 minutes, room temperature), the supernatant is recovered, purified water (50 mL) and saturated saline solution (50 mL) are added, mixed well, and placed in a 250 mL centrifuge bottle and centrifuged. Separation (4500 rpm, 4 minutes, room temperature) separated the aqueous phase and removed it. This operation was repeated several times, and the neutrality of the aqueous phase was confirmed with a pH test paper.
By the above centrifugation operation, the liquid became three phases. Although the intermediate phase was dark red, the β-cryptoxanthin concentration was confirmed by HPLC, and the content was about 35% of the uppermost hexane phase.
The hexane phase and the dark red intermediate phase were recovered and concentrated with care not to cause bumping on a rotary evaporator to obtain 3.71 g of an oil. This oily substance was suspended in a hexane: acetone = 90%: 10% mixed solution (40 mL), mixed vigorously and then centrifuged (4500 rpm, 4 minutes, room temperature), and the supernatant was collected.
Silica gel (Merck Japan Co., Ltd., silica gel 60, 400 g) is suspended in a hexane: acetone = 90%: 10% mixed solution and then packed into a glass chromatographic column, and the recovered material is gently placed on the top of the silica gel in the column. And elution was performed with a mixture of hexane: acetone = 90%: 10%.
Since β-cryptoxanthin corresponds to the thickest red band part, this part was fractionated and concentrated by a rotary evaporator to obtain 0.136 g of concentrate.
Ethanol was added thereto, and the mixture was heated and dissolved at about 60 ° C., and when left in a cool dark place, dark purple crystals having a metallic luster were deposited.
By filtering this, 0.104 g of high-purity free β-cryptoxanthin was obtained. This product showed no impurities other than carotenoids by NMR, and was 96% pure by HPLC analysis.

With reference to FIG. 16, each value which concerns on the carotenoid composition for every process which concerns on Example 4 is demonstrated.
In Example 4 of FIG. 16, the low-concentration carotenoid composition had a β-cryptoxanthin content (as a free form) of about 1.3% and a recovery rate from the raw material of 87% or more.
The medium-concentration carotenoid composition had a β-cryptoxanthin content (as a free form) of about 2.13% and a recovery rate of about 83%.
Moreover, about the high concentration carotenoid composition recrystallized by the ethanol treatment after the silica gel chromatographic separation of the organic phase obtained after alkali hydrolysis, the β-cryptoxanthin content (as a free form) is almost 100%, The recovery rate was 61% or more.
In this way, free β-cryptoxanthin can be produced with high efficiency by performing simple silica gel chromatographic separation only once using the peel of Citrus unshiu.

[Production of highly concentrated carotenoid composition using oysters]
(Example 5)
Next, an example in which a carotenoid composition having an increased concentration is produced using persimmon (Diospyros kaki Thunb.) Peel will be described with reference to FIG. The production process was the same as in Example 1 described above, and an oily substance containing carotenoid was finally obtained.
FIG. 17 shows an example of producing a carotenoid composition using 74.34 g of dried oyster peel. Oysters contain lutein, zeaxanthin, β-cryptoxanthin, and β-carotene as the main carotenoids. Therefore, in FIG. 17, among the compositions in each step, in each fraction, the content (mg) related to the total of recovery rate (%), lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and carotenoid. , Content (%) is shown.
In Example 5 of FIG. 17, the total carotenoid content as a free form in the raw material was 0.035%, and the content in the hexane extract was about 4.1%.
The total carotenoid content in the hexane-insoluble part was 7.5% or more, but the recovery rate from the raw material was about 26%.
The low-concentration carotenoid composition had a total carotenoid content (as a free form) of about 3.6% and a recovery rate from the raw material of 73% or more.
The medium-concentration carotenoid composition produced based on the low-concentration carotenoid composition has a total carotenoid content (as a free form) of about 8.9%, a recovery rate from the raw material of 70% or more, and does not require a high concentration. In some cases, the recovery rate was the best.
The high-concentration carotenoid composition produced based on the medium-concentration carotenoid composition had a total carotenoid content (as a free form) of about 28.3% and a recovery rate from the raw material of 52% or more.

In Example 5, the concentration ratio of carotenoid in the high-concentration carotenoid composition compared to the hexane extract was lutein 6.4 times, zeaxanthin 7.1 times, β-cryptoxanthin 7.0 times, β-carotene 4.6. It was twice.
As described above, although there are some differences depending on the carotenoid species, they all show almost the same tendency, and even the raw materials having a composition containing carotenoids other than β-cryptoxanthin can be concentrated in this embodiment. It shows that there is.

[Production results of high-concentration free carotenoid composition derived from oyster peel]
In Example 5, the high-concentration carotenoid composition (38 mg) obtained from oyster peel was dissolved in t-methylbutyl ether (3 mL), 10% potassium hydroxide methanol solution (2 mL) was added, and the mixture was replaced with nitrogen. Then, it was allowed to stand overnight in a dark place at room temperature for hydrolysis.
Transfer the hydrolysis reaction liquid to a separatory funnel, add t-butyl methyl ether as appropriate, add water and stir, wash the organic phase with water, centrifuge in a 50 mL centrifuge tube (4500 rpm, 4 min) to separate the aqueous phase. Removed. This operation was repeated several times, and the organic phase was recovered when the water washing solution became neutral. The collected organic layer was concentrated with a rotary evaporator, hexane was added and dispersed by sonication. Thereafter, a dark purple precipitate having a metallic luster was obtained on standing. This was collected by filtration to obtain a high-concentration free carotenoid composition.

Here, with reference to FIG. 18, the yield, content, and content rate of the free carotenoid composition obtained by hydrolysis of the high concentration carotenoid composition derived from oyster peel will be described.
Referring to the content of the insoluble part in FIG. 18, the high concentration free carotenoid derived from oyster peel of Example 5 was analyzed by HPLC. As a result, 17.9% lutein, 31.4% zeaxanthin, and 40-cryptoxanthin 40. A mixture of 6% and β-carotene 1.5% carotenoid content of 90% or more could be obtained.
Moreover, when converted from this content rate, about 95% of the carotenoid contained in the high concentration ester type carotenoid composition of the raw material could be recovered.
As described above, a carotenoid composition with high efficiency and increased concentration can be obtained from oyster peel using the method for extracting a carotenoid composition according to the embodiment of the present invention.

Thus, according to this example, a carotenoid composition containing ester-type β-cryptoxanthin at a high concentration and a high-purity free β-cryptoxanthin crystal can be obtained from Satsuma mandarin. In addition, in other mandarin oranges, such as citrus oranges and ponkan, and citrus fruits, a composition containing a high concentration of β-cryptoxanthin and free β-cryptoxanthin crystals can be obtained by the same process.
Further, a carotenoid composition containing a high concentration of ester-type β-cryptoxanthin and a crystal consisting only of a carotenoid containing free β-cryptoxanthin can be obtained from oysters.

[Production of highly concentrated carotenoid composition using loquat and papaya]
In addition, regarding loquat and papaya, it is also possible to obtain a crystal composed of only a carotenoid composition containing ester-type β-cryptoxanthin at a high concentration and a carotenoid containing free β-cryptoxanthin.

  Note that the configuration and operation of the above-described embodiment are examples, and it is needless to say that the configuration and operation can be appropriately changed and executed without departing from the gist of the present invention.

  The method for producing a composition containing a high concentration of ester-type β-cryptoxanthin according to an embodiment of the present invention effectively uses citrus fruit juice and fruit peels produced during canning, and various forms of pulp produced during fruit production. High-concentration β-cryptoxanthin ester-type composition and high-purity free β-cryptoxanthin that can be used efficiently as pharmaceuticals, health supplements, quasi drugs, and food additives Is possible.

Claims (11)

Add hexane to the powder dried to 5% or less of the moisture content of the plant containing carotenoids to obtain a hexane extract in the soluble part,
Concentrate the hexane extract to obtain a concentrate,
After re-dissolving by adding a redissolved solution to the concentrate, cooling to about −20 ° C. to −30 ° C., collecting the soluble part by centrifugation, and concentrating to obtain a low-concentration carotenoid composition,
A predetermined amount of ethanol is added to the low-concentration carotenoid composition, cooled to about −20 ° C. to −30 ° C., and centrifuged to obtain an insoluble part ,
The plant body is one of mandarin oranges, oysters (Diospyros kaki Thunb.), Loquat (Eriobotrya japonica (Thunb.) Lindl.), Or papaya (Carica papaya L.) . Production method. The powder is a powder obtained by drying the peel of mandarin oranges,
The concentrate is a residue obtained by adding 10% to 20% ethanol: 80 to 90% water to the hexane extract and distilling under reduced pressure while heating at 50 ° C to 60 ° C. And
The redissolved solution added to the concentrate is hexane about 3 times the weight of the concentrate,
The ethanol predetermined amount added to the low concentration carotenoid composition, method for producing a carotenoid composition as claimed in claim 1, wherein the ethanol of about 3 to 4 times the weight of the low-concentration carotenoid composition . The powder is a powder obtained from pulp of a site originating from citrus sand,
The redissolved solution added to the concentrate is hexane about 3 times the weight of the concentrate,
The ethanol predetermined amount added to the low concentration carotenoid composition, method for producing a carotenoid composition as claimed in claim 1, wherein the ethanol of about 3 times the weight of the low-concentration carotenoid composition. The powder is a powder obtained from a pulp obtained by collecting diatomaceous earth adsorbed insoluble matter generated in the filtration process step during the production of citrus clear fruit juice,
The redissolved solution added to the concentrate is hexane about 3 times the weight of the concentrate,
The method for producing a carotenoid composition according to claim 1 or 2, wherein the predetermined amount of ethanol added to the low-concentration carotenoid composition is about three times the weight of the low-concentration carotenoid composition. . In the carotenoid composition obtained in claim 2 ,
Add about 3 to 4 times the amount of hexane / ethanol mixture containing 40% hexane and 60% ethanol at a volume ratio, cool to -20 ° C to -30 ° C, centrifuge, collect the insoluble part , and reduce the pressure. A method for producing a high-concentration carotenoid composition comprising concentrating. In the carotenoid composition obtained in claim 3 or 4 ,
Add about 3 to 4 times the amount of hexane / ethanol mixture containing 40% hexane and 60% ethanol at a volume ratio, cool to -20 ° C to -30 ° C, perform centrifugation, and recover the insoluble part. A method for producing a high-concentration carotenoid composition. Hydrolyzing the high-concentration carotenoid composition obtained in claim 5 ;
After removing the alkali by washing with water, a predetermined amount of hexane is added to the residue obtained by concentrating the organic phase,
The insoluble part obtained by dispersing and leaving by ultrasonic treatment is collected by filtration,
A method for producing a high-purity free carotenoid composition, wherein ethanol is added to the insoluble part and dispersed by ultrasonic treatment, and the insoluble part produced by standing is recovered by filtration. Hydrolyzing the high-concentration carotenoid composition obtained in claim 6 ;
After removing the alkali by washing with water, a predetermined amount of hexane is added to the residue obtained by concentrating the organic phase,
After dispersion by sonication, the insoluble part obtained by allowing to stand is recovered by filtration,
A method for producing a high-purity free carotenoid composition, wherein ethanol is added to the insoluble part and dispersed by ultrasonic treatment, and the insoluble part produced by standing is recovered by filtration. Hydrolyzing the high-concentration carotenoid composition obtained in claim 6 ;
After removing the alkali by washing with water, the hexane phase and the dark red intermediate phase are recovered and concentrated,
Hexane 85% to 95%: Suspended in a hexane / acetone mixture containing 5% to 15% acetone, centrifuged to collect the supernatant,
The supernatant is eluted with the hexane / acetone mixture by column chromatography using silica gel,
A method for producing a high-purity free carotenoid composition characterized by fractionating and collecting the thickest red band part. In the carotenoid composition obtained in claim 1 ,
Add about 3 to 4 times the amount of hexane / ethanol mixed solution containing 40% hexane and 60% ethanol by volume ratio, cool to -20 ° C to -30 ° C, centrifuge, collect the soluble part,
The method for producing a high-concentration carotenoid composition, wherein the powder is obtained by drying oyster skin . Hydrolyzing the high concentration carotenoid composition obtained in claim 10 ;
After removing the alkali by washing with water, a predetermined amount of hexane is added to the residue obtained by concentrating the organic phase,
The insoluble part obtained by dispersing and leaving by ultrasonic treatment is collected by filtration,
A method for producing a high-purity free carotenoid composition, wherein ethanol is added to the insoluble part and dispersed by ultrasonic treatment, and the insoluble part produced by standing is recovered by filtration.

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