JP6458314B2 - Method for producing ceramide - Google Patents

Description

  The present invention relates to a method for producing sphingolipids by koji mold culture using shochu as a culture medium.

  A large amount of shochu is by-produced in the production of shochu using yeast and yeast as microorganisms. This shochu is composed of a lot of liquid (about 90% by weight or more) and a small amount of solid, and the water contains amino acids, organic acids, partially unfermented sugar, vitamins, ash, fermented metabolites, etc. ing. The solid content contains cells such as koji molds and yeasts as well as fibers such as cellulose and pectin derived from cereals as insoluble components. As described above, shochu is easily rotted due to its rich nutritional components, and has a high BOD (biological oxygen demand) and SS (suspended material). For this reason, when discarding shochu, methane fermentation treatment is performed, the concentration of organic substances in the liquid is reduced, solid-liquid separation is performed to remove sludge, and the liquid content is aerobic biological treatment (active It is necessary to purify by the sludge method.

  For this reason, for example, after solid-liquid separation of shochu, the solid content is sent to a fermentor and fermented with koji mold to liquefy the solid content (solubilization) and reduce the volume. A method has been proposed in which a lactic acid bacterium is added to a storage tank so as to suppress the generation of spoilage and malodor (Patent Document 1).

  On the other hand, since shochu contains various functional components, there are attempts to collect and effectively use the functional components contained in shochu. For example, inoculating koji mold into shochu and cultivating it, decomposing / solubilizing the solid content in the koji mold with an enzyme produced by koji mold, etc. It has been proposed that the liquid component can be used for feed, food materials, seasonings, and the like (Patent Document 2).

  As another attempt to utilize the functional components contained in shochu, a liquid ethanol extract or extraction residue obtained by separating shochu by a method such as centrifugation is added to a pharmaceutical composition or health food, and this ethanol extraction It is disclosed that the liquid or the extraction residue is effective for the prevention and treatment of cell proliferative diseases such as tumors (Patent Document 3).

  Moreover, paying attention to the sphingolipid contained in shochu, a method of using shochu as a raw material for collecting it is also known. More specifically, the free ceramide content is characterized by removing impurities from the alcohol extract of the fermented fermenter, which is a by-product generated during the production of the fermented product using koji, using a non-solvent of free ceramide. Many methods for obtaining sphingolipids have been proposed (Patent Document 4). In addition, a method for producing sphingolipids including a step of extracting sphingolipids from shochu from which moisture has been removed has been proposed (Patent Document 5).

  Here, the sphingolipid is composed of a sphingoglycolipid in which a long chain fatty acid, sphingosine, which is a long chain fatty acid and an acid amide bond, has a common structure and a sugar is glycoside bonded thereto, and a sphingophospholipid in which a phosphate and a base are bound. It is a generic name for various compounds that are classified, and is distributed mainly in biological membranes of microorganisms and animals and plants.

  Some of these compounds have been shown to have bioactive functions. In particular, ceramide (or “free ceramide”) is present as a component of human skin organs and plays a role in preventing water loss or skin dryness. Incidentally, when the amount of ceramide produced decreases with age, it becomes easy to develop dry skin, atopic dermatitis and the like.

  About ceramide, it has been reported that there are skin beautifying effects such as moisturizing, improvement of rough skin prevention, and improvement of wrinkle prevention (Non-Patent Documents 1 and 2). It has also been reported that glycosphingolipids having a specific structure extracted from sponges are effective as antitumor agents and immunostimulators (Patent Document 6). In addition, glycosylceramide having a specific structure has been shown to be effective as a therapeutic agent or an abortive agent for autoimmune diseases (Patent Document 7).

  Since the sphingolipid contained in the above shochu is insoluble in water, it hardly exists in the liquid content of the shochu and exists in its solid content. For this reason, in the method shown in Patent Document 5, in order to recover the sphingolipid contained in the shochu, after removing the water by solid-liquid separation in advance, the organic matter is used for the solid content of the sphingolipid. Extraction is performed. In this case, since the liquid component occupying about 90% or more of the shochu is not used at all and becomes a large amount of waste liquid, it must be purified at a great cost.

  On the other hand, since many sphingolipids are present in biological membranes of animals and plants or microorganisms, animal-derived sphingolipids can also be extracted from bovine brain and the like. However, human use of animal-derived sphingolipids such as bovine brain by oral ingestion tends to be avoided due to the BSE problem and the like. In addition, plant-derived sphingolipids are extracted from grains such as wheat and rice, beans such as soybeans, and fruits such as pineapples, but because the content of sphingolipids in these plant materials is small ( For example, see Patent Document 4), which has a problem in terms of manufacturing cost.

  In order to solve these problems, a method for producing sphingolipids using microorganisms has also been proposed. For example, microorganisms such as acetic acid bacteria (Patent Document 9), bacteria belonging to the genus Pseudomonas (Patent Document 10), yeast (Patent Document 11) are cultured to proliferate the cells, and sphingolipids are collected from the collected cells. The method is known.

  However, in the conventional production method using these microorganisms, a) an expensive medium is required for culturing the microorganism, b) the cell growth rate is low in the cultivation of acetic acid bacteria, and c) bacteria. When using microorganisms such as yeast, there are problems in terms of safety and management of microorganisms.

JP 2008-126187 A JP-A-6-315369 Patent No. 4071062 JP2012-41518 JP 2012-228246 A Patent No. 3068810 JP 2004-131481 A JP 2012-228246 A JP 2007-306882 A Japanese Patent Laid-Open No. 10-81655 Special table 2008-518612

Fragrance Journal, Volume 23, p. 81-89, 1995 Bioindustry, Vol. 19, p. 16-26, 2002

  For this reason, development of a technology that can efficiently produce sphingolipids having high added value by a safer method using a cheaper medium is eagerly desired, but it has not yet been developed. .

  Therefore, the main object of the present invention is to provide a method for producing sphingolipid more efficiently.

  As a result of intensive studies in view of the problems of the prior art, the present inventor has found that the above object can be achieved by employing a specific processing step, and has completed the present invention.

That is, the present invention relates to the following method for producing ceramide .
1. A method for producing ceramide , comprising:
(1) the liquid fraction is 90 wt% or more, steps in the liquid content of 100 wt% of the SDB and the remainder is a solid as medium or at least one nutrient source of carbon and nitrogen sources in the liquid, Used as a medium by adding
(2) a culture process for culturing koji molds in the medium,
(3) A recovery process for recovering the cultured koji molds,
(4) A method for producing ceramide , comprising a collecting step of collecting ceramide from the collected cells of Aspergillus oryzae.
2. Item 2. The production method according to Item 1, wherein in the culturing step, culturing is performed under a condition in which a nutrient source is not additionally supplied to the medium from the outside.
3. The production method according to claim 2, wherein the nutrient source is at least a carbon source.
4). Item 4. The production method according to any one of Items 1 to 3, wherein the koji mold is at least one selected from the genus Aspergillus.
5. Item 5. The production method according to any one of Items 1 to 4, wherein the medium is a liquid medium.

  According to the production method of the present invention, sphingolipid (particularly ceramide) can be produced more efficiently. In particular, since a cheap and safe liquid component of shochu is used as a culture medium for koji mold, the resulting sphingolipid is highly safe and can be efficiently produced at a relatively low cost. At the same time, the liquid content of shochu, which is a by-product of shochu production, can be used as an inexpensive and economical medium for koji molds. As a result, high-value-added alternatives to shochu methane fermentation or feed / fertilizer applications It can be used and can contribute to environmental conservation.

  The sphingolipid thus obtained (sphingolipid containing ceramide) can be used, for example, as a raw material (additive) for functional foods, cosmetics, pharmaceuticals and the like.

It is a graph which shows the dry weight of the microbial cell in each culture medium after culture | cultivation. It is the chromatogram obtained by analyzing the free ceramide extracted from the microbial cell of Aspergillus by TLC. It is the chromatogram obtained by analyzing the free ceramide extracted from the microbial cell of Aspergillus by TLC. It is a graph which shows content of the free ceramide extracted from the microbial cell in each culture medium. It is a graph which shows the relationship between an aspergillus culture and culture | cultivation temperature. It is a graph which shows the relationship between the dry weight of the collect | recovered microbial cell, and culture | cultivation time.

The production method of the present invention is a method of producing a sphingolipid,
(1) a culture process for culturing koji molds in a medium containing a liquid component of shochu,
(2) A recovery process for recovering the cultured koji molds,
(3) It includes a collecting step of collecting sphingolipids from the collected gonococcal cells.

Culture process In the culture process, koji molds are cultured in a medium containing a liquid component of shochu.

  The shochu used here is not particularly limited, and shochu produced in a general manufacturing process of shochu can be used. In general, shochu is produced by adding koji mold to koji raw material. More specifically, water and yeast are added to the koji and allowed to lay for 1 to about 30 days to prepare the primary mash. Next, raw materials such as cereals and potatoes and water are added, and the mixture is further laid for 1 to about 30 days, followed by secondary charging to make secondary moromi. Subsequently, the secondary moromi is distilled in a single manner to make the original sake. Here, the shochu is mainly a residue of secondary moromi distilled in the distillation step. In the production method of the present invention, the shochu generated in the distillation process of shochu production can be suitably used.

  In general, about 90% by weight or more of the shochu is liquid (moisture or the like) and the rest is solid. In the present invention, the above-mentioned liquid is used as a culture medium for koji molds. More sphingolipids can be produced by using the liquid component. In addition, by using the liquid component, it is possible to more efficiently perform the cell collection operation in the subsequent recovery process.

  In the present invention, the liquid content of shochu can be used as it is as a medium, but a nutrient source can be added as necessary within the range not impairing the effects of the present invention. That is, the culture medium in the present invention includes a culture medium in which a nutrient source is added to the liquid content of shochu as well as a culture medium having a liquid content of shochu of 100% by weight. Examples of the nutrient source to be added include at least one of a carbon source and a nitrogen source. Examples of the carbon source include saccharides such as glucose. Examples of the nitrogen source include various amino acids.

  In particular, in the culturing process of the present invention, it is desirable to perform culturing under conditions in which no nutrient source is additionally supplied to the medium from the outside. Therefore, for example, a medium having a liquid content of shochu of 99 to 100% by weight can be suitably used. By using such a medium, a koji mold having a high concentration of sphingolipid can be obtained, and as a result, sphingolipid can be produced in a higher yield.

  The medium may be either solid or liquid, but is preferably a liquid medium from the viewpoint of the efficiency of the separation / purification process. That is, in the present invention, it is desirable to culture the koji mold by liquid culture. However, solid content may exist in the liquid medium within a range that does not hinder the effects of the present invention.

The koji mold that can be used in the culturing step is not particularly limited as long as it can produce sphingolipids, but those belonging to the genus Aspergillus can be preferably used. Examples of such koji molds include the following.
a) Aspergillus kawachii
b) Aspergillus luchuensis
c) Aspergillus usami
d) Aspergillus saitoi
e) Aspergillus oryzae

  In the culturing process of the present invention, at least one of these koji molds can be used. In particular, at least one of Aspergillus kawachi and Aspergillus oryzae is preferable in terms of safety or ease of management of microorganisms. .

  The culture temperature of the koji mold in the culturing step is usually 30 to 40 ° C, and particularly preferably 30 to 35 ° C. Since the temperature of the koji is generally 32 to 37 ° C., the culture is not usually performed at a temperature higher than this. In addition, the culture time may be appropriately set according to the yield of the desired sphingolipid, usually within a range of 24 to 150 hours. As long as the nutrient source is present in the medium, the koji mold grows, but in the liquid culture, the cells are in the form of beads, so the cells inside the beads may die due to insufficient supply of oxygen or nutrient sources. Therefore, the upper limit of the culture time is preferably about 150 hours, particularly about 110 hours.

In the recovery step , the cultured koji molds are recovered.

  The recovery method is not particularly limited, and may be performed, for example, according to a general solid-liquid separation method. As the solid-liquid separation method, a known method such as filtration or centrifugation can be employed.

  It is desirable to subject the microbial cells to a drying treatment in advance in order to remove water prior to the collection step after collecting the gonococcal cells. As a drying method, for example, a technique such as freeze drying or vacuum drying can be applied.

Collection process In the collection process, sphingolipids are collected from the collected gonococcal cells.

  The method for collecting sphingolipids from Aspergillus oryzae cells is not particularly limited, and for example, a method of extracting sphingolipids from Aspergillus oryzae cells using a solvent can be suitably employed. More specifically, there may be mentioned a method comprising a step of dissolving the sphingolipid by mixing the cells of Aspergillus and the solvent and then removing the solvent to obtain the sphingolipid.

  About selection of a solvent, it is desirable that it is a highly polar substance (polar solvent). Examples thereof include a) at least one polar solvent or b) a mixed solution of at least one polar solvent and a nonpolar solvent.

  Examples of the polar solvent include organic chlorine compounds such as chloroform, alcohols such as methanol and ethanol, and acetone. In particular, when extracting sphingolipids for use in foods and the like, ethanol, acetone and the like designated as food additives are suitable. Moreover, as a nonpolar solvent, normal-hexane etc. which are designated by the food additive are mentioned.

  At the time of extraction, various contaminants other than sphingolipids are also extracted at the same time. Therefore, in order to obtain highly pure sphingolipids, a process of removing contaminants before and after extraction (preferably before extraction) is performed. It is preferable to implement. For example, lipids such as triglyceride contained in the microbial cells are cleaved in advance with a potassium hydroxide-methanol solution and decomposed into glycerin and fatty acids. At this time, the sphingolipid is not decomposed because there is no ester bond. Then, after the treatment with the potassium hydroxide-methanol solution, chloroform and water are added to dissolve glycerin and fatty acid, which are decomposition products of lipids such as triglyceride, in the aqueous phase, while sphingolipid is dissolved in the chloroform phase. In this way, lipids such as triglyceride contaminants can be removed. In addition, as another method for removing impurities, the sphingolipid can be extracted and then purified using various techniques such as known chromatography.

  Analysis of sphingolipids after extraction of sphingolipids from bacterial cells is performed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC evaporative light scattering detection method), mass spectrometry (LC / MS, GC / MS), etc. as appropriate. Can be combined.

  The features of the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the examples. In the examples, “%” indicates “% by weight”.

Examples 1-2
(1) Preparation of medium A supernatant was obtained by centrifuging shochu shochu at 7000 rpm for 10 minutes. The obtained supernatant was filtered through a cellulose mixed ester membrane filter having a pore size of 0.45 μm to remove solids contained in the liquid. 100 mL of the obtained filtrate (liquid content) was dispensed into a conical flask with a baffle (capacity 500 mL) and sterilized by an autoclave.

(2) Culture of Aspergillus Next, spores of Aspergillus kawachii were added to the sterilized filtrate. More specifically, white spore spores dispersed in a 0.05% Triton X aqueous solution sterilized in advance were added so that the spore concentration was 8.0 × 10 ⁵ cells / 100 mL. Then, it culture | cultivated by the shaking culture method using the shaking culture machine (The Taitec Co., Ltd. make, Bio Shaker BR-300LF type | mold). The culture conditions were culture time: maximum 88 hours, culture temperature: 30 ° C., and shaking speed of the incubator: 100 / min.

  For comparison, a medium having the composition shown in Table 1 was used as an artificial medium (Comparative Example 1). Moreover, liquid culture of Aspergillus was performed under the same conditions as described above using a medium in which glucose was added to the above shochu filtrate to a concentration of 2% (Example 2). (A) Artificial medium (Comparative Example 1), (b) Shochu filtrate (Example 1), (c) Shochu filtrate added with 2% glucose (Example 2), which are the above three types of media In each medium, the total nitrogen amount (TN), which is one of the indicators of nutrient sources, was analyzed using a total nitrogen analyzer (manufactured by Actac Co., Ltd., Super Kel 1500 type). As a result, 0.071% was obtained in (a) and 0.080% in (b) and (c), both of which were almost the same. Note that the sugar (glucose) concentrations in the above (a) and (b) were both set to 2%.

(3) Weight measurement of bacterial cells After culture, the cells were filtered using a hydrophilic membrane-treated PTFE (polytetrafluoroethylene) membrane filter (pore size: 1 μm), and the collected bacterial cells were transferred to an eggplant-shaped flask (capacity: 500 mL). After freeze-drying, the weight of the obtained dried cells was measured. As a result, Comparative Example 1 (artificial medium) was 0.20 g, Example 1 (filtration) was 0.58 g, and Example 2 (filtration + 2% glucose) was 0.77 g. These results are also shown in FIG. In addition, a microbial cell weight is a value per 100 mL of culture media.

  As is clear from the results in FIG. 1, it can be seen that the weight of the bacterial cells in Example 1 is larger than that in Comparative Example 1 and is about three times that of Comparative Example 1.

  The total nitrogen amount in Comparative Example 1 and Example 1 is almost the same value, whereas glucose, which is one of typical carbon sources, is added in Comparative Example 1 at 2%, whereas Example 1 Since it is after alcohol fermentation, it contains almost no sugar. Nevertheless, as for the gonococcus, as compared with Comparative Example 1, Example 2 resulted in more microbial cells growing. Thus, it can be inferred that the medium of Example 1 contains some component that promotes the growth of Aspergillus, and it can be seen from this Example that the liquid content of shochu is suitable as the medium of Aspergillus.

  Moreover, when the culture medium of Example 2 which added 2% of glucose to the shochu filtrate was used, the cell weight further increased as compared with the culture medium of Comparative Example 1 and Example 1. Although Comparative Example 1 and Example 2 have almost the same total nitrogen amount and glucose concentration, it can be said that Example 2 has reached about 4 times the bacterial yield, which is a result supporting the above-mentioned assumption.

(3) Measurement of content of ceramide Next, the content of free ceramide contained in the recovered gonococcal cells was quantified by thin layer chromatography (TLC) according to the following method. First, 20 mL of a chloroform / methanol (volume ratio of 1: 1) mixed solvent was added per 1 g of microbial cells, and ultrasonic waves were irradiated in a hot water bath at about 40 ° C. for 1 hour. Furthermore, 20 mL of 0.8 M potassium hydroxide-methanol solution was added, and ultrasonic waves were irradiated for 1 hour in a hot water bath at about 40 ° C. to decompose lipids such as triglyceride, which are contaminants in the cells. Subsequently, 50 mL of chloroform and 23 mL of water were added and the mixture was separated into two liquids. The lower layer (chloroform / methanol layer) was separated, and the mixed organic solvent of chloroform / methanol was removed with an evaporator. To the resulting dried product, a mixed solvent of chloroform / methanol (volume ratio 1: 1) was added to make up to 5 mL, and the free ceramide was quantitatively analyzed by thin layer chromatography (TLC). The analyzer used was an automatic thin layer detector Iatroscan (Iatroscan ^new MK-5, manufactured by Yatron Co., Ltd.). In the analysis, Chromarod-S4 manufactured by Mitsubishi Chemical Medience Corporation was used, and chloroform / methanol / water (volume ratio 90: 10: 1) was used as a developing solvent. C18 Ceramide (Cat. # C263050) manufactured by Toronto Research Chemicals Co. Ltd. was used as a standard substance for free ceramide.

  In this way, free ceramide, which is one of the sphingolipids extracted from gonococcal cells, was analyzed by TLC. The chromatogram at that time is shown in FIG. FIG. 3 shows a chromatogram of the standard substance (ceramide) used in the quantitative analysis. Moreover, the ratio (%) of the weight of free ceramide per unit weight of the dried microbial cells was 0.34% in Comparative Example 1, 1.09% in Example 1, and 0.67% in Example 2. there were. These are also shown in FIG.

  As is clear from the results of FIG. 4, it can be seen that the content of free ceramide in Example 1 reaches about 3 times that of the artificial medium of Comparative Example 1. This result shows that the shochu liquor is extremely effective in collecting the sphingolipids from the cells by using the shochu liquor as a medium for koji mold.

  Moreover, in the culture medium of Example 2 to which 2% of glucose was added, the free ceramide content of the microbial cells increased as compared with the case of Comparative Example 1, but it can be seen that the free ceramide content is lower than that of Example 1. This result suggests that the sphingolipid content in the microbial cells increases in an environment in which gonococcus is less likely to grow (for example, under conditions where glucose as a carbon source is not added).

(4) Relationship between Aspergillus Culture and Culture Temperature FIG. 5 shows the relationship between Aspergillus culture and culture temperature. As is clear from the results of FIG. 5, in the present invention, 30 to 35 ° C. is considered a suitable temperature for culture.

(5) Relationship between Aspergillus culture and culture time FIG. 6 shows the relationship between the dry weight of the collected cells and the culture time. As is apparent from the results of FIG. 6, the growth rate of Aspergillus gradually increased after about 24 hours from the start of the culture. In addition, the cell weight increased with the culture time.

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

Claims (5)

A method for producing ceramide , comprising:
(1) the liquid fraction is 90 wt% or more, steps in the liquid content of 100 wt% of the SDB and the remainder is a solid as medium or at least one nutrient source of carbon and nitrogen sources in the liquid, Used as a medium by adding
(2) a culture process for culturing koji molds in the medium,
(3) A recovery process for recovering the cultured koji molds,
(4) A method for producing ceramide , comprising a collecting step of collecting ceramide from the collected cells of Aspergillus oryzae. The production method according to claim 1, wherein in the culturing step, culturing is performed under a condition in which no nutrient source is additionally supplied to the medium from the outside. The production method according to claim 2, wherein the nutrient source is at least a carbon source. The production method according to any one of claims 1 to 3, wherein the koji mold is at least one selected from the genus Aspergillus. The manufacturing method in any one of Claims 1-4 whose said culture medium is a liquid culture medium.