JP6085847B2 - Method for producing sphingolipid - Google Patents

Description

  The present invention relates to a method for producing sphingolipids from shochu.

Sphingolipids are a group of lipids having a sphingoid base, which is a long-chain amino alcohol, as a common component. Ceramide, a kind of sphingolipid, exists as a major lipid component in the stratum corneum of the skin and is known to perform a moisturizing function. Since ceramide is deficient with age, there is a great demand in the field of skin care cosmetics for the purpose of supplementation.
Sphingolipids are abundant in brain white matter and have been mainly collected from bovine brain until now, but it has become difficult to separate from bovine brain due to the occurrence of mad cow disease, and now there are new sources derived from plants or microorganisms. It has been demanded.

As new sources of sphingolipids, grains such as yeast, rice, soybeans and wheat have already been used. In addition, a technique for extracting ceramide, a kind of sphingolipid from konjac (Non-patent Document 1: A rapid method of total lipid extraction and purification. BLIGH EG, DYER WJ. Can J Biochem Physiol. 1959 Aug; 37 (8): 911-7.), Or a method for producing sphingolipids from sake koji and miso bacteria Aspergillus oryzae, Aspergillus sojae has also been developed (Patent Document 1: JP 2010-154788, Patent Document 2: JP 2010-41972 gazette).
However, the presence of sphingolipids from shochu containing grains and yeasts is not clear.
In addition, a method is known in which after extracting sphingolipids using chloroform / methanol, the extract is washed with high-pressure carbon dioxide (Patent Document 3: Japanese Patent Application Laid-Open No. 2003-119492). Also known is a method for increasing the amount of sphingolipid by increasing the humidity and increasing the amount of sphingolipid (Patent Document 4: JP 2010-22279 A).
However, in the conventional method, there is a limit to the organic solvent to be used when producing sphingolipids for edible use, and more expensive equipment is required for processing the extracted sphingolipids. Development of a method for producing sphingolipids by a technique has been desired.

  On the other hand, hundreds of thousands of tons of shochu are generated annually in Kyushu. Up to now, shochu has been dumped into the ocean, but due to the increasing international interest in environmental issues, shochu from being dumped into the ocean has been banned along with the revision of the Marine Pollution Control Law. Yes.

JP 2010-154788 JP 2010-41972 JP 2003-119492 A JP 2010-22279

A rapid method of total lipid extraction and purification.BLIGH EG, DYER WJ. Can J Biochem Physiol. 1959 Aug; 37 (8): 911-7.

  An object of this invention is to provide the method of manufacturing a sphingolipid efficiently from shochu.

As a result of intensive studies to solve the above problems, the present inventor has found that a large amount of sphingolipid is contained in the shochu, and has completed the present invention.
That is, the present invention is as follows.
(1) A method for producing sphingolipid, comprising a step of extracting sphingolipid from shochu from which moisture has been removed.
(2) The method according to (1), wherein the shochu is produced in the production process of shochu using shochu as a raw material, and (3) the cereal is rice or barley the method of.
(4) Aspergillus kawachii, Aspergillus saitoi, Aspergillus awamori, Aspergillus nakazawai, Aspergillus uslusi, spermus luslus, i (Aspergillus luchensis) and Aspergillus niger, and at least one selected from the group consisting of a microorganism belonging to the genus Rhizopus, a microorganism belonging to the genus Monuscus and a microorganism belonging to the genus Penicillium, Method.
(5) A method for producing sphingolipid, comprising a step of culturing shochu bacteria, removing water from the culture, and collecting sphingolipid from the obtained water-removed product.
(6) Aspergillus kawachii, Aspergillus saitoi, Aspergillus awamori, Aspergillus nakazawai, Aspergillus gususi, Aspergillus sus (Aspergillus luchensis) and Aspergillus niger, and at least one selected from the group consisting of a microorganism belonging to the genus Rhizopus, a microorganism belonging to the genus Monuscus and a microorganism belonging to the genus Penicillium, Method.
(7) The method according to (5) or (6), wherein the culture temperature is 30 to 50 ° C.
(8) The method according to any one of (5) to (7), wherein the culture method is liquid culture or solid culture.

According to the present invention, a method for producing a sphingolipid is provided.
According to the method of the present invention, a sphingolipid different from the conventionally known sphingolipid can be extracted from the shochu. In the present invention, new sphingolipids can be produced simply by culturing shochu bacteria. According to the present invention, it is possible to extract sphingolipids using ethanol that is inexpensive and harmless to the human body, and further, it is possible to increase the production amount of sphingolipids by culturing koji molds at a high temperature. it can.

It is a figure which shows the position of the sphingolipid contained in a shochu sphingolipid. It is a figure which shows the comparison with another sphingolipid source. It is a figure which shows the origin of shochu sphingolipid. It is a figure which shows the result of having extracted sphingolipid from the culture of Aspergillus. It is a figure which shows the result of having extracted the sphingolipid except moisture.

Hereinafter, the present invention will be described in detail.
1. Outline The present invention has been completed based on the finding that a novel sphingolipid is contained in shochu, and is characterized by extracting sphingolipid from the shochu. In addition, the present invention is characterized in that sphingolipids are extracted from the culture (from which water has been removed) by culturing shochu bacteria.
In the present invention, sphingolipids can be extracted using a predetermined organic solvent. At this time, water is removed from the shochu raw material. That is, water is removed from the shochu raw material by centrifugation or the like, and the sphingolipid is extracted from the remaining solid portion.
Moreover, a novel sphingolipid can be produced by shochu. Furthermore, it was also clarified that this novel sphingolipid is derived from shochu.
In the present invention, when producing sphingolipids from koji molds, koji molds are cultured at 30 ° C. to 50 ° C., preferably 30 to 45 ° C., more preferably 40 ° C. Generally, since the temperature of the koji temperature is 32 to 37 ° C., the culture is not usually performed at a temperature higher than this. On the other hand, in the present invention, it can be cultured at a normal culture temperature, but it can be efficiently cultured by culturing at 38 ° C to 50 ° C, preferably 38 ° C to 45 ° C, more preferably 40 ° C. It became possible to produce. According to the method of the present invention, for example, 4 mg of sphingolipid per unit raw material can be obtained by extracting the sphingolipid after removing water from the culture obtained by culturing at 40 ° C. This amount is 1.2 to 1.5 times the amount obtained by the conventional method, which shows that the method of the present invention is extremely excellent.

No research examples have been found that have analyzed whether sphingolipids are contained in shochu. Therefore, in the present invention, detection of sphingolipids in shochu and evaluation / analysis of the amount thereof were carried out as part of measures for utilizing shochu.
Sphingolipids were extracted from shochu according to a conventional method and then developed by silica gel thin layer chromatography for detection. As a result, it was possible to detect spots at positions similar to the purified bovine brain cerebroside used as a marker (Fig. 1). Next, the amount was evaluated, and it was clarified that 4.2-6.3 mg of sphingolipid was contained in 1 g of shochu as a dry weight. This result revealed for the first time that sphingolipids were contained in shochu.

2. Production of Sphingolipid from Shochu The raw material used in the method of the present invention is not particularly limited as long as it is a cereal or root vegetable that can be made into a koji by growing a koji mold, such as rice, barley, wheat, sweet potato, Brown sugar, buckwheat, baron, etc. can be used. It is particularly preferable to use barley.

  The sphingolipids to be manufactured are classified into sphingophospholipids in which a ceramide having a fatty acid and an acid amide bond bonded to a sphingoid has a common structure and a sugar is glycoside bonded thereto, and a sphingophospholipid in which a phosphate and a base are bonded. In the present invention, the sphingolipid is not particularly limited, and examples thereof include ceramide, sugar-linked sphingolipid, sphingophospholipid, and sphingosine base, and ceramide is preferable.

The koji mold that can be used in the method of the present invention is not particularly limited as long as it can produce sphingolipid, and includes microorganisms belonging to the genus Aspergillus.
For example, Aspergillus kawachii, Aspergillus saitoi, Aspergillus awamori, Aspergillus nakazawai, Aspergillus usgilis, Aspergillus usgisis Examples include Aspergillus niger.
In addition, the microorganisms that can be used in the present invention are not limited to microorganisms belonging to the genus Aspergillus, but other genera such as microorganisms belonging to the genus Rhizopus, microorganisms belonging to the genus Monuscus, microorganisms belonging to the genus Penicillium, etc. Can do. The microorganisms belonging to these genera are exemplified below.
Microorganisms belonging to the genus Rhizopus: Rhizopus oryzae, Rhizopus oligosporus, Rhizopus niveus, Rhizopus microspores, Ronzo pus Microbes belonging to the genus Monascus: Monuscus anka, Monuscus purpureus, Monuscus ruber, Monuscus pilosus, Monuscus aurantiacus ), Monuscus kaoliang, etc. Microbes belonging to the genus Penicillium: Penicillium camemberti, Penicillium roqueforti, Penicillium -Graucum (Penicillium glaucum), Penicillium caseicolumn (Penicillium caseicolum), etc. In the present invention, these koji molds can be used singly or in combination, but Aspergillus kawachi is preferred.

Specifically, the following commercially available products and deposited microorganisms can be used.
A. kawachii: NRIB SC60
A. saitoi: NRIB2601
A. awamori: NIRB2013
A. nakazawai: NRIB2623
A. usamii: NIRB2004
Since the microorganism belonging to the genus Rhizopus, the microorganism belonging to the genus Monuscus and the microorganism belonging to the genus Penicillium have the characteristics of a fungus that grows in a mycelium type used in the production of foods, in the present invention as well as the above Aspergillus genus Can be used.

Shochu is produced by the following process. That is, the rice that is the raw material for shochu lees is washed, soaked in water, and steamed. The koji mold is added to this steamed rice to produce koji. The koji mixes water and yeast and lays it for about 1 to 30 days to make a primary charge. This makes primary moromi. Next, raw cereals (barley, wheat, rice, rice bran, etc.) and water are mixed with the primary mash and further laid for 1 to 30 days for secondary charging. Thereby, secondary mash is manufactured. As the raw material to be mixed with the primary mash, wheat is used when producing wheat shochu, and rice is used when producing rice shochu. Subsequently, the secondary mash is distilled through a single distiller. In this distillation step, raw alcohol is produced by evaporating the alcohol and then cooling it back to a liquid.
The raw sake obtained by distillation is purified by filtration and stored in barrels or bottles.
In the above process, shochu is the remainder of the secondary moromi distilled in the distillation process.
In the present invention, the shochu generated in the distillation process during the manufacturing process of shochu can be used.

Shochu contains a lot of water, and the cocoon is suspended in water or is in a water-containing state with a content rate of 30 to 95%. Therefore, in the present invention, moisture is substantially removed from the shochu containing a large amount of moisture. “Removing moisture” means a state in which some moisture may be contained if the shochu is not suspended in water, a state in which the shochu is in a paste state, It means that the powder is in a dry state, that is, the water content contained in the shochu is 20% or less. The method for removing moisture is not particularly limited, and examples thereof include centrifugation and filtration. Centrifugation is preferable in terms of simple operation. Centrifugation conditions are 10 to 10,000 × g, preferably 3,000 × g for 1 to 50 minutes, preferably 5 minutes.
In this way, water is substantially removed from the shochu and a precipitate is obtained.
The above shochu may be dried as it is or as a precipitate by heating, spray drying, freeze drying or drying under reduced pressure. In the present invention, the shochu from which water has been substantially removed is also referred to as a “dried product”.
Extraction of sphingolipids from the shochu is performed as follows by dissolving the dried product of the shochu in an extraction solvent.
The extraction solvent is not particularly limited as long as it is used for lipid extraction, and examples thereof include chloroform / methanol extract and ethanol. When extracting sphingolipids for food, it is preferable to extract with ethanol or the like. Thereafter, the organic layer is removed to obtain an extracted lipid.

As an analysis method of the obtained extract, for example, thin layer chromatography can be mentioned. Sphingolipids can be analyzed as a control since there are commercial standards such as glucosylceramide. Using a commercially available silica gel thin layer plate, development is performed using a solvent such as chloroform-methanol, and color is developed using an orcinol reagent. Thereby, the qualitative analysis of the sphingolipid in an extract can be performed. Quantitative analysis and structural analysis of sphingolipid can be performed by appropriately combining high performance liquid chromatography and mass spectrometry (LC / MS, GC / MS, HPLC light scattering detection method).
Further, the obtained glycosphingolipid composition can be used as it is by solidifying and powdering it using a method such as freeze-drying or spray-drying.
The sphingolipid obtained by the method of the present invention (extraction from shochu) is ceramide, sugar-linked sphingolipid, sphingophospholipid, sphingosine base and the like, and the yield is 2 to 8 mg per 1 g of the raw material.

3. Production of sphingolipids from Aspergillus in the present invention The present invention also provides a method for producing sphingolipids characterized by culturing shochu bacteria, removing water from the resulting culture, and then collecting the sphingolipids from the water-removed product To do. “Culture” means any of the cultured gonococcal cells, culture supernatant, and crushed cells.
As for the koji mold, the microorganisms shown below can be used singly or in appropriate combination as described above.
Microorganisms belonging to the genus Aspergillus: Aspergillus kawachii, Aspergillus saitoi, Aspergillus awamori, Aspergillus usperi, Aspergillus usperi, Aspergillus usperi・ Aspergillus luchensis, Aspergillus niger, etc. Microbes belonging to the genus Rhizopus: Rhizopus oryzae, Rhizopus oligosporus, Rhizopus rizopus, Rhizopus Microbes belonging to the genus Monuscus, such as Rhizopus microspores and Rhizopus stolonifer: Monuscus anka, Monascus Microbes belonging to the genus Penicillium such as Monuscus purpureus, Monuscus ruber, Monuscus pilosus, Monuscus aurantiacus, Monuscus kaoliang, etc.・ Penicillium camemberti, Penicillium roqueforti, Penicillium glaucum, Penicillium caseicolum, etc.

As the culture of koji mold, either solid culture or liquid culture can be adopted.
In the case of solid culture, steamed rice, steamed barley, or the like is used as the medium, and the culture temperature is 20 to 50 ° C. for 1 to 5 days. In the present invention, the culture temperature is preferably 30 ° C to 50 ° C, more preferably 30 ° C to 45 ° C, and most preferably 40 ° C. The culture period when the culture temperature is 40 ° C. is preferably 1 to 20 days.
For liquid culture, modified Czapek-Dox medium (3% soluble starch or glucose, 0.1% NaNO ₃ , 0.05% KCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ / 7H ₂ O, 0.001% FeSO ₄ , pH 6.0) and the like, and culture at a temperature of 20 to 50 ° C. for 1 to 5 days. In the present invention, the culture temperature is preferably 20 ° C to 50 ° C, more preferably 35 ° C to 45 ° C, and most preferably 40 ° C. The culture period when the culture temperature is 40 ° C. is preferably 1 to 20 days.
Subsequently, sphingolipids are extracted from the culture and collected. At that time, particularly in the case of liquid culture, the culture is concentrated as appropriate by centrifugation or filtration. Thereby, a moisture-removed product (dried product) from which the moisture has been substantially removed from the culture can be obtained.
Analysis of sphingolipids after extraction is performed by appropriately combining thin layer chromatography, high performance liquid chromatography and mass spectrometry (LC / MS, GC / MS, CE-MS MS, HPLC light scattering detection method) as described above. be able to.
Examples of the sphingolipid obtained by the method of the present invention (culture of Aspergillus) include ceramide, sugar-linked sphingolipid, sphingophospholipid, sphingosine base, and the like, and the yield is 1 to 10 mg per 1 g of the raw material.

4). Sphingolipids and uses thereof Sphingolipids obtained by the method of the present invention can be used as additives for foods, drinks, cosmetics or moisturizers, and are added or blended after the production process or production of foods, drinks, cosmetics or moisturizers. Can be obtained.
Such foods and drinks are provided as health foods, functional foods, foods for specified health use, dietary supplements, and the like.

The beverage is not particularly limited, and examples thereof include tea, black tea, oolong tea, coffee, juice, sports beverage, milk, yogurt and the like.
The food is not particularly limited. For example, noodles (udon, soba, etc.), breads, confectionery (cookies, snacks, rice crackers, chocolate, ice cream, pudding, jelly, rice cake, gum, etc.), dairy products (cheese) Etc.), frozen foods, fish paste products (ham, sausage, kamaboko, etc.), seasonings (miso, soy sauce, dressing, mayonnaise, etc.), various prepared vegetables and the like.
However, the food and drink are not limited to the above-described examples.

Cosmetics include foundations, makeup cosmetics, packs, creams (cold cream, night cream, burnishing cream, hyzenic cream, nutritional cream, hormone cream, cleansing cream, hand cream, lip balm, etc.), milky lotion, lotion Etc.
The amount of sphingolipid to be included in the food / beverage products and cosmetics is not particularly limited, but is, for example, 1 μg to 100 μg, preferably 30 μg to 80 μg, more preferably 60 μg per 300 mg.
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Barley shochu was used as a sample.
A 0.3 g sample (water content: 0%) from which moisture was removed (dried) by heating the shochu at 100 ° C. was obtained. Extract this sample with 2.0ml chloroform / methanol (1: 1), mix with 2.0ml 0.8M KOH-methanol, incubate at 42 ℃ for 30 minutes, and then mix with 5ml chloroform and 2.25ml distilled water. Centrifugation was performed at 2000 rpm for 5 min to take the lower organic phase, which was used as the extracted lipid.
The extracted sphingolipid was developed with Silica gel TLC and detected with orcinol-sulfate. To confirm the position, purified bovine brain cerebroside, a kind of glycosphingolipid, was used.
As a result, it was possible to detect a spot at almost the same position as cerebroside (Fig. 1), and the resulting sphingolipid was HPLC (Inertsil SIL 100A 5 μm, 4.6 (250 mm; GL science Inc, Japan) chloroform: 95% Purified with methanol (0min 100: 0, 15min 75:25, 20min 10:90, 40min 100: 0), and the major peaks were analyzed using Bruker ESI-MS / MS m / z (736.6) and MS / MS (162.0 , 254.3, 416.3, 416.3), it was identified as D-Glucosyl-b-1,1'-N-2-hydroxylpalmitoyl-4,8-sphingadienine, which is one of cerebrosides.

Next, when the amount was evaluated, it was clarified that 4.2 to 6.3 mg of sphingolipid was contained in 1 g of shochu as a dry weight.
The results revealed for the first time that shochu contains sphingolipids, and that it contains more sphingolipids than so far reported sphingolipid sources such as soybean (Fig. 2). ).

  The sphingolipid content of soybean currently used as a sphingolipid source is about 1.7 mg / g (corresponding to cerebroside, Vesper et al., J. Nutrition, 1239-1250, 1999). Therefore, shochu contains about 3.1 times the amount of sphingolipids contained in soybean, indicating that shochu is a suitable source of sphingolipids.

In this example, the search for a lipid source in barley shochu was conducted using barley, wheat straw and shochu yeast, which are raw materials for barley shochu.
Sphingolipids were extracted from dried samples of barley, wheat straw and shochu yeast (water content: 0%) in the same manner as in Example 1, and developed and detected. In addition, when producing wheat straw, white birch (Aspergillus kawachii) used for shochu production was used, and association shochu yeast 2 was used as shochu yeast.
The results are shown in FIG.
From FIG. 3, it was found that the sphingolipid found in the shochu was cerebroside and was mainly derived from Aspergillus.

In this example, sphingolipids were produced by culturing shochu bacteria.
Rice or wheat was allowed to absorb 30% of its weight, steamed for 30 minutes, allowed to cool to room temperature, inoculated with Aspergillus kawachii, and cultured at 30 ° C. for 3 days.
After the culture, the culture was centrifuged (3000 × g, 5 minutes), and the precipitate was heated at 100 ° C. to remove moisture and dried. Extract 0.3g of dried sample (water content: 0%) with 2.0ml of chloroform / methanol (1: 1), mix with 2.0ml of 0.8M KOH-methanol, incubate at 42 ℃ for 30 minutes, and further 5ml Chloroform and 2.25 ml of distilled water were mixed and centrifuged at 2000 rpm for 5 min to take the organic phase of the lower layer, which was used as the extracted lipid.
The extracted sphingolipid was developed with Silica gel TLC and detected with orcinol-sulfate.
To confirm the position, purified bovine brain cerebroside, a kind of glycosphingolipid, was used.
As a result, a spot could be detected at a position similar to cerebroside (FIG. 4), and the collected sphingolipid was found to be cerebroside.

In this example, barley shochu was used as the sample.
The shochu was separated into liquid and solid by centrifugation (3000 rpm, 5 min), and the water content was reduced to 20%. The solid portion was then stirred for 5 minutes with ethanol and left at room temperature for 1 day. The ethanol content was dried and subjected to TLC analysis.
The results are shown in FIG.
In FIG. 5, lane 4 is obtained by removing water and then extracting with ethanol to dry the ethanol portion, and lane “barley shochu lees” is extracted with chloroform / methanol. A band corresponding to cerebroside was observed near the Rf value of 5.5 (the part surrounded by a red circle).
The result of FIG. 5 shows that sphingolipid can be extracted efficiently by removing water from the shochu and extracting with ethanol.

  The present invention is useful for the production of sphingolipids having uses as foods and drinks, cosmetics or moisturizers.

Claims (7)

Cultivated shochu fungus without mixing yeast, removed water from the culture, and obtained water-removed product Extracted fromIncluding sphingolipidA composition containingManufacturing method. Shochu fungi are Aspergillus kawachii, Aspergillus saitoi, Aspergillus awamori, Aspergillus nakazawai, Aspergillus usamiru, i ) And Aspergillus niger, and a microorganism belonging to the genus Rhizopus, a microorganism belonging to the genus Monuscus, and a microorganism belonging to the genus Penicillium. The method according to claim 1 or 2, wherein the culture temperature is 30 to 50 ° C. The method according to any one of claims 1 to 3, wherein the culture method is liquid culture or solid culture. Cultivated shochu fungus without mixing yeast, removed water from the culture, and obtained water-removed product Extracted fromSphingolipid produced by a method comprising the step of:A composition comprisingUse for cosmetics. Cultivated shochu fungus without mixing yeast, removed water from the culture, and obtained water-removed product Extract fromSphingolipid produced by a method comprising the stepsA composition comprising The composition according to claim 6, which is a cosmetic.