WO2012062043A1 - Method for producing promoter from ceramide and/or glucosylceramide - Google Patents

Abstract

A method for producing promoter from ceramide and/or glucosylceramide comprises culturing yeasts in a culture medium containing bean dregs and carbohydrates, and obtaining the promoters produced by ceramide and/or glucosylceramide from the supernatant thereof. The method can utilize available substances as the culture medium and use the yeast to obtain the promoters produced by ceramide and/or glucosylceramide cheaply and efficiently.

Description

 Method for producing ceramide and/or glucosylceramide production promoter

 The present invention relates to a method for producing a neuronal amide production promoting agent and/or a glucosylceramide production promoting agent capable of promoting skin production of ceramide and/or glucosylceramide. More particularly, the present invention relates to the production of a ceramide and/or glucosylceramide production promoter capable of promoting skin production of ceramide and/or glucosylceramide by using a specific microorganism, a by-product okara of a food processing industry or the like as a main raw material of a culture medium. method. Background technique

 The skin has the ability to produce ceramide and glucosylceramide. Ceramide synthesized by epidermal cells is secreted in the skin, especially in the cuticle of the epidermis, and is a major component of intercellular lipids. Ceramide, one of the sphingolipids, is present in the stratum corneum of the skin and accounts for about 50% of the intercellular lipids in the stratum corneum. Sphingolipid is a generic term for a complex lipid having a sphingosine skeleton. Glucosylceramide is a sphingolipid that combines glucose with ceramide. The ceramide synthesized by epidermal cells is temporarily stored in the form of glucosylceramide or sphingomyelin; it is then excreted outside the cell, in glucocerebrosidase and neurophospholipidase (sphingomyelinase). Under the action of ceramide, ceramide is formed again, thereby functioning as an intercellular lipid. Ceramide and glucosylceramide in the skin have physiological effects such as moisturizing function of the skin and barrier function of the skin.

 It has been known that sphingolipids such as ceramide, glucosylceramide, and galactosylceramide have effects of promoting the retention of water in the stratum corneum and improving skin roughness (Patent Documents 1 to 2). Although the skin has the ability to produce ceramide and glucosylceramide, the skin tends to become dry due to environmental, climatic, and the like. Therefore, it is not sufficient to produce ceramide by the skin itself to improve the moisturizing function of the skin.

Therefore, people have tried a method of supplementing ceramide to the skin from the outside. For example, Patent Document 3 describes a method for producing glucosylceramide by culturing Candida tropicalis (Owifefe tropicalis) JPCCY0004 strain (NITE P-570) by using Candida (0«3⁄43⁄4) It can be applied to the dry skin of the human body to improve the skin. Skin moisturizing function. However, this method is not a method for ceramide and/or glucosylceramide production promoter, but merely obtains a large amount of glucosylceramide present directly from the cultured cells, and thus the nerve of the synthesized glucosylceramide The amide skeleton is different from the ceramide skeleton of ceramides present in the human body, and has carbon-carbon double bonds at two places and has a branch. Therefore, when such glucosylceramide directly extracted from microorganisms is applied to human skin, there is a possibility that it cannot be adapted to human skin and causes problems in use safety.

 In addition, the method of supplementing ceramide to the skin from the outside has a problem that the moisturizing effect is insufficient in persistence as in the conventionally used moisturizing agent, and the skin in some cases is insufficiently absorbed by the externally supplied ceramide or the like, thereby failing to be performed. Give full play to your moisturizing effect.

 On the other hand, if it can enhance or promote the ceramide or glucosylceramide production ability of the skin itself, it is not necessary to externally supplement ceramide or glucosylceramide, and it is possible to produce ceramide and/or glucosylceramide by promoting the skin itself. To improve the barrier function and moisturizing function of the skin. Thus, since ceramide or glucosylceramide is produced by the skin itself, ceramide or the like having a conventional structure of the skin is synthesized, and thus there is no safety problem such as poor adaptability to the skin.

 Therefore, research on substances which promote the production of epidermal ceramides and the like in the stratum corneum of the skin has recently become quite popular. Among them, as a substance capable of promoting the production of ceramide, a ceramide production promoter having an extract of Ganoderma lucidum, Bee Pollen, and Chuanxiong as an active ingredient has been reported (Patent Documents 4 to 6). However, since these ceramide production promoters are all transferred from plant raw materials, the extraction operation takes a long time, and there are limitations in the availability of natural resources, so that the manufacturing cost thereof is high.

Further, Patent Document 7 discloses a ceramide synthesis promoter capable of activating ceramide synthesis of epidermal cells themselves in the surface layer of the skin, which comprises a bacterium culture containing nicotinic acid and/or nicotinamide as an active ingredient. The culture of the bacterium may be a lactic acid bacteria culture, a bifidobacterium culture, a lentinus bacillus culture or a yeast culture. However, as can be seen from the examples of Patent Document 7, the ceramide production amount of the lactic acid bacteria culture or the yeast culture (Comparative Example ~ 4) containing no nicotinic acid and/or nicotinamide was not significantly changed from the control group. Therefore, the ceramide synthesis promoter substantially contains nicotinic acid and/or nicotinamide as an active ingredient, and the effect of promoting ceramide synthesis is not sufficient, and there is still room for improvement. Moreover, since the medium used in the preparation of the accelerator substantially contains nicotinic acid and/or nicotinamide as an active ingredient, the preparation cost of the ceramide synthesis accelerator is high.

 In addition, yeast is a single-celled fungus that is widely distributed in nature and is mainly grown in a slightly acidic, sugary environment. Can survive in an oxygen-deficient environment. Can be used for brewing production. There are currently more than 1000 kinds of yeasts, including yeasts of the genus iSchizosaccharomyces, yeast of the genus Pichia, Candida, yeast of Debaryomyces, and iCryptococcus. Hamenu yeast, Bueux sinensis BuUera yeast, Rhodotorula ί Rhodotoruki yeast, Saccharomyces sp. (Sporobolomyces yeast, Saccharomyces cerevisiae <iBrettanomyces> yeast, Yarrowia (mwa) yeast Wait.

 Schizosaccharomyces Schi noodles ccharomyces yeast has long been used in food processing, which produces ethanol and is also used as a research material for biology.

 Further, the Pichia genus yeast has the following properties, uses, and the like.

 Pichia cell lines are spherical, elliptical, elongated, occasionally conical, but do not form a apex. Asexual reproduction is a multilateral budding, and some species can form spores. Each ascus usually contains 1 to 4 ascospores, occasionally more than 4. The spores are cap-shaped, hemispherical or spherical. When the ascus is mature, it usually splits, a few do not split, and the sacs are joined or not joined to form an ascus, which occurs between the mother cell and the bud or between two independent cells. Both hyphae and pseudohyphae can be used as ascospores, but the morphology does not become large or become spindle-shaped, with the same or different combinations; can be assimilated nitrate, DBB reaction is negative.

 The taxonomic status of Pichia is: Ascomycetes (o? >^to), Semi-Ascomycetes {Hemiascomycetes, Saccharomycetales, Saccharomycetaceae, Pichia (Combies czz') o).

 The use of Pichia iPichia is, for example, the use of methanol to produce useful substances such as antibiotics, biological proteins, etc.; or it can also be used as genetically engineered bacteria. Pichia pastoris is used to produce reduced glutathione, and other Pichia pastoris are used to produce some proteins/enzymes.

In addition, it is known that Candida (Ccmdick's microorganisms usually have the following uses: for example, Candida crudea (Candida valida is used to produce lipase, Candida tropicalis Candidametapsilosis, Candida utilis (cmdida utilis, etc.) produce Brewing, glycerin, edible yeast, organic acids, and enzyme preparations can also be used in the word industry; and, the microorganisms of Candida Ccmdida can also be used to treat waste from industrial and agricultural by-product processing industries, and to produce edible proteins.

 Further, it is known that the microorganism of Debaryomyces generally has the following industrial uses, for example, fermenting glucose to produce D-arabitol, or converting xylose into xylitol.

 In addition, some studies have been conducted on the microorganisms of Cryptococcus. For example, it has been reported in the literature that the C. serrata variant produces β-galactosidase. In addition, it has also been found that the strain of Cryptococc laurentii) has an inhibitory effect on the storage of citrus acid rot (Geotrichum citri-aurantii).

 In addition, it is known that microorganisms of the genus Hansenula ficm la are capable of producing ethyl acetate, thereby increasing the flavor of the product, and thus are generally used in the wine making and food industries.

 In addition, it is known that microorganisms of the genus Bulrush, Bi le, are generally used for food fermentation, such as sausage fermentation.

 In addition, some studies have been conducted on the microorganisms of Rhodotorula. For example, certain species of the genus Rhodotorula have weak oxidation of hydrocarbons and can synthesize beta-carotene. For example, the sticky red yeast variety can oxidize terpene to produce fat, and the content can reach 50~60% of dry biomass. Under certain conditions, a-alanine and glutamic acid can also be produced, and the ability to produce methionine is also strong, up to 1% of dry biomass.

 Further, it is known that microorganisms of the sporozoite sporobolomyces are useful in industries such as accumulation of coenzyme Q10 in bacteria. In addition, the microbes of Sporobo myces are also attracting attention as fungi for carotenoids, L-carnitine and other useful substances.

 In addition, it is known that the microorganism of the genus Brettanomyces belongs to yeast, which is widely found in nature and usually multiplies in barrel storage to produce ethyl phenol. Therefore, in the wine industry, it is generally unpopular as a contaminating bacteria that destroys the aroma of beer, wine, and the like.

 In addition, Yarrowia, a microorganism such as rra 'a lipolytica, is known to be particularly useful for the production of polyunsaturated fatty acids (PUFAs).

However, there have been no reports on the use of yeasts (especially the yeasts of the above-mentioned genus) to prepare ceramide production promoters or glucosylceramide production promoters. Patent Document 1 Japanese Patent Laid-Open No. 61-260008

 Patent Document 2: Japanese Unexamined Patent Publication No. 61-271205

 Patent Document 3: JP-A-2010-22217

 Patent Document 4: Japanese Special Report No. 2005-194240

 Patent Literature 曰本特开 2010-70499号

 Patent Document 6: Japanese Special Feature No. 2010-150237

 Patent Document 7: Japanese Patent Publication No. 9-194383 SUMMARY OF THE INVENTION

 The present invention has been made to solve the above-mentioned problems in the prior art, and an object thereof is to provide a method for producing a ceramide production promoter and/or a glucosylceramide production promoter at low cost and high efficiency.

 The present inventors conducted intensive studies to solve the above-described technical problems, and as a result, found that: culturing yeast in a medium containing okara and sugar can obtain a ceramide production promoter and/or a glucosylceramide production promoter at low cost and high efficiency. The invention has thus been completed.

 The present invention provides a method for producing a ceramide production promoter and/or a glucosylceramide production promoter, wherein a yeast is cultured in a medium containing okara and a saccharide, and a ceramide production promoter is obtained from the supernatant thereof. And / or glucosylceramide production enhancer.

 The present invention also relates to the use of a fermented extract of bean dregs for promoting the production of ceramide and/or glucose amide, which is obtained by culturing yeast in a medium containing okara.

 The present invention also relates to the use of a fermented extract of bean dregs as a ceramide production promoter and/or a glucosylceramide production promoter, which is obtained by culturing yeast in a medium containing soybean dregs.

 The present invention also relates to the use of a fermented extract of bean dregs for producing a ceramide production promoter and/or a glucosylceramide production promoter, wherein the fermented extract of the bean dregs is obtained by culturing yeast in a medium containing okara.

In the present invention, the yeast may be selected from the group consisting of the yeast of the genus Chinosaccharomyces, the yeast of the genus iPichia, the yeast of Candida ί Can Canida, the yeast of Debaryomyces, the yeast of Cryptococcus i. Saccharomyces (Hans Let La yeast, B. ssp. Bid ra) Yeast, Rhodotorula yeast, Saccharomyces sp. iSporobolomyces yeast, Brettmomyces yeast, Yarrow (rraw) ^) At least any one of the yeasts.

 According to the production method of the present invention, a specific microorganism can be used and a readily available substance such as bean dregs can be used as a main raw material of the medium, whereby a ceramide production promoter and/or a glucosylceramide production promoter can be obtained inexpensively and efficiently. The accelerator can effectively improve the skin's ability to produce ceramide/glucose ceramide, thereby effectively improving the skin's moisturizing ability. detailed description

 In the production method of the present invention, yeast is cultured in a medium containing okara and saccharides, and a ceramide production promoter and/or a glucosylceramide production promoter is obtained from the supernatant.

 (Micro-organisms that can be used in ceramide and/or glucosylceramide production promoters)

 The microorganism which can be used in the production of the ceramide and/or glucosylceramide production promoter of the present invention may be selected from the group consisting of Schizosaccharomyces yeast, Pichia yeast, Candida iCandidO yeast, Debali. Saccharomyces cerevisiae (Debaryomyces yeast, Cryptocaryon genus (Cr_y;? tococc) Yeast, Hansenula Η Η n n n n 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 n n n n n n n n n n n n n n n n n n n n n n n n n n n n And at least any one of Brettanomyces yeast and Yarrowia (wrov a) yeast.

 Examples of the microorganism of the Schizophyllum haromyces which can be used in the present invention include Schizosaccharomyces octo?on«, Schizosaccharomyces pombe, and the like which are commonly used in the food industry. Also included are variants of these microorganisms obtained according to the usual methods.

Among them, Schizosaccharomyces octoporus is preferred from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting effect. Examples of the microorganism of the Pichia genus Picic0 which can be used in the present invention include the Pichia pastoris Pichi, which is commonly used in the food industry, and Pichia guilliermondii 5|5 Pichia ( Pichia norvegensis Pichia membranifaciens ί Pichia burtonii), Pichia farinose, Pichia Kluyveri, etc.; also includes microbes obtained according to the usual methods Mutant strain.

 Among them, from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting effect, Pichia anomala is preferred, Pichia guilliermondii, Norwegian Bi Pichia norvegensis ^ Pichia membranifaciens Pichia burtonii.

 Among them, Pichia membranifaciens is also known as Pichia membranaefaciens, Pi Pilot alcoholophila,

 (Pichia hyalospora), Pichia scaptomyzae^ Willia belgica or Zygopichia guilliermondii

Dematium chodati 、柯达氏拟内抱霉 ( Endomycopsis chodati) 隐伯顿毕赤酵母 Hyphopichia burtonii) 或贝雷丝孢酵母（ T chosporon behrendi 。 Pichia burtonii) Also known as Candida (Candida)

Dematium chodati, Endomycopsis chodati (Hyphopichia burtonii) or T. chosporon behrendi.

As a microorganism of Candida which can be used in the present invention, candida candida (Candida _Z ey noid _eS , Candida boidinii, near smoothing) used in the food field can be cited. Candida parapsilosis, Ccmdida magnoliae, Candida maltosa, Candida steatolyticcd, Candida butyri, Candida rhagi , at least one microorganism selected from Candida metapsilosis, Candida albicans, Cmdida holmii, Candida valida, etc.; The obtained mutant strains of these microorganisms. These microorganisms function as follows, and by culturing in a medium containing okara and saccharides, ceramide and/or glucosylceramide which enhance epidermal cells can be obtained from the supernatant thereof. a substance that produces capacity. Among them, the product obtained has a higher ceramide From the viewpoints of an amine production promoting effect and/or a glucosylceramide production promoting effect, Candida zeylanoides, Candida boidinii, Candida parapsilosis, Magnolia pseudoacacia Candida magnoliae, Candida maltosa, Candida steatolytica (synonym/previous name: Candida hellenical), Candida butyr (Candida butyr , Candida rhagii or Candida metapsilosis.

Examples of the microorganism of the genus Debcuyomyces which can be used in the present invention include Debaryomyces hameniO and Deb ^^ co/jwo^ which are used from the food field. /n^ At least one microorganism selected from the yeast Debaryomyces vanrijiae, etc.; and a mutant strain of these microorganisms obtained according to a usual method. These microorganisms function as follows, by culturing in the presence of okara and sugar The medium is cultured, whereby a substance for increasing the ceramide and/or glucosylceramide production ability of the epidermal cells can be obtained from the supernatant thereof, wherein the obtained product has a higher ceramide production promoting effect and/or glucose. ceramide production promoting effect viewpoint, it is preferable Debaryomyces hansenii (Debaryomyces hansenii), or yeast Fanlideba Wo ¹ J (Debaryomyces vanrijiae)

 Examples of the microorganism of the cryptococcal yptococcus which can be used in the present invention include Cryptococcus hungaric sinensis, Cryptococcus hungaric s, Cryptococcus terreus, and the like; These include mutant strains of these microorganisms obtained according to the usual methods.

Wherein the product obtained from the viewpoint of having higher ceramide production promoting effect and / or glucose ceramide production promoting effect, preferably Cryptococcus laurentii (Cryptococcus laurentii) ¹ J Wo Hungary or Cryptococcus (Cryptococcus Hungaricus).

Examples of the microorganism of the genus Hansenula which can be used in the present invention include Ham ula polymorpha and Hansenula sinensis (Ha Song mda ciferrii, sub-membrane Han) used from the food field. "Shen Yeast Hansenu subpelliculosa" ^ at least one microorganism selected from Hansenu satumus (iHansenula anomala), etc.; Method for obtaining mutant strains of these microorganisms. These microorganisms function as follows, and by culturing in a medium containing okara and saccharides, a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant. Among them, from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting-promoting effect, Hansenula polymorpha or Schwannella Ha-resolved La cifetrif) Among them, the homologous/previous name of Hansenula polymorpha): Pichia angusta, Hansemda angusta, polymorpha Ogataea polymorpha ).

 As a microorganism of the S. sphaeroides Bulk which can be used in the present invention, the spruce-sucking yeast Bt m tsugae used in the food field can be cited. Polygonum buckwheat (Bu!lera Cao iabilis) At least one microorganism selected from the genus Bullera albus, etc.; and a mutant strain of these microorganisms obtained according to a usual method. These microorganisms function as follows, in a medium containing okara and sugar By culturing, a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant thereof, wherein the obtained product has a higher ceramide production promoting effect and/or glucosylceramide. From the viewpoint of producing a promoting effect, Spirogymus sinensis iBulkm tsugae (synonym/previous name: Sporobolomyces tsugae) is preferred.

 Examples of the microorganism of Rhodotorchus Rhocto which can be used in the present invention include Rhodotola mucilaginosa, Rhodotorula glutinis, Rhodotorula minuta, and Rhodotorula pallida. , Rhodotorula rubra, etc.; also includes variants of these microorganisms obtained according to the usual methods.

 Among them, Rhodotorula mucilaginosa or Rhodotorula glutinis is preferred from the viewpoint that the obtained product has a higher ceramide production promoting effect and/or a glucose ceramide production promoting effect.

As a spore-type iSp that can be used in the present invention, b. The microorganism of hmy _CeS may, for example, list at least one microorganism selected from the group consisting of Spowbohmyces roseus, the genus Sporobolomyces salmonicolor, or the porphyry sporobolomyces ruber; According to A variant of these microorganisms obtained by the usual method. These microorganisms function as follows, and by culturing in a medium containing okara and saccharides, a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant. Among them, from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting effect, Sporozolios roseus is preferred.

 The microorganism of the genus Brett momyces which can be used in the present invention is selected from the group consisting of Brettanomyces claussenii or Brettanomyces anomalus used in the food field. At least one microorganism; and a variant of these microorganisms obtained according to a usual method. These microorganisms function as follows, and are cultured in a medium containing okara and saccharides, whereby substances capable of increasing ceramide and/or glucosylamide production ability of epidermal cells can be obtained from the supernatant. Among them, Brettanomyces claussenii is preferred from the viewpoint that the obtained product has a higher ceramide production promoting effect and/or a glucosylceramide production promoting effect.

As Yarrow Saccharomyces can be used in the present invention (RV «) microorganisms include yeast Yarrow lipolytica (Y _ar that ia lipolytic ^ or the like according to conventional method yeast Yarrow lipolytica obtained (Yarn ia lipofytica Such mutants of microorganisms. These microorganisms function as follows, and by culturing in a medium containing okara and saccharides, ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant thereof. Among them, from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting effect, Yarrowia lipolytic^ is preferred, and its synonym/has The names are: Caw3⁄4⁄4⁄4 lipolytica, (Candida paralipolytica), Saccharomycopsis lipolytica., Endomycopsis lipolytica, Candida albicans (Pseudomonilia deformans).

 The microorganisms (yeast) to which the present invention relates can be purchased from commercial sources such as the China General Microorganisms Collection and Management Center (CGMCC), the China Center for Type Culture Collection (CCTCC), and the Industrial Microbial Resources and Information Center (CICIM) of Jiangnan University.

In the production method of the present invention, one type of the above microorganism (yeast) may be used alone, or two or more types of the above microorganisms (yeast) may be used in combination. (Method for producing ceramide and/or glucosylceramide production promoter) The method for producing a ceramide and/or glucosylceramide production promoter of the present invention comprises culturing yeast in a medium containing okara and saccharides, and A ceramide and/or glucosylceramide production promoter is obtained in the supernatant. The specific steps of the method are as follows.

 <Medium>

 In the present invention, it is preferred to culture one or more of the above microorganisms in a liquid medium.

 The medium used in the present invention is a medium containing bean dregs and sugar as a main component. Both the okara and the saccharide are easily available, and thus, the medium of the present invention has an advantage of being low in cost. Further, in addition to this, for example, 1% peptone, 0.5% yeast powder or the like may be added as appropriate.

 Among them, the bean dregs refers to a substance obtained by immersing soybeans in water to sufficiently absorb water, pulverizing, filtering, removing water, and draining. It is also possible to use soybean residue remaining in the soybean product manufacturing process such as pressing soybean milk in the food industry.

 The saccharide may be glucose, sucrose or maltose, etc., and they may be used singly or in combination. Further, from the viewpoints of improving the productivity and utilization of the fermentation, and the effect of promoting the ceramide production promoting effect and the glucosylceramide production promoting effect, glucose is preferred.

 The medium of the present invention is preferably composed of okara, glucose, and water from the viewpoint of enhancing the ceramide production-promoting effect and the glucosylceramide production-promoting effect, and more preferably the content of the bean dregs in the medium is 5 to 20 w/v%, more preferably 8 to 12 w/v%, the content of glucose is 0.2 to 2 w/v%, more preferably 0.8 to 1.2 w/v%.

 <Cultivation conditions>

The cells of the above microorganisms are directly inoculated into a ceramide/glucose ceramide production promoter production medium. As a culture condition for producing a ceramide and/or a glucosylceramide production promoter, the physiological characteristics of the yeast microorganism, the temperature and time necessary for optimizing the growth of the yeast microorganism, and the ceramide production promoting effect and/or glucose From the viewpoint of the ceramide production promoting effect, it is preferably cultured at a culture temperature of 20 to 40 ° C, more preferably 27 to 35 ° C for 1 to 7 days, preferably for 48 to 96 hours, more preferably for 68 to 76 hours. . <Recovery and Purification of Ceramide and/or Glucose Ceramide Production Accelerator> From the viewpoint of obtaining a more pure product, it is preferred to further purify the culture obtained by the above culture to obtain a supernatant. The purification method can be carried out by a combination of a usual operation such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, crystallization, or the like as needed. The supernatant is preferably obtained by centrifugation. The speed of the centrifugation is preferably from 2000 to 4000 rpm, more preferably from 2500 to 3500 rpm, and the centrifugation time is preferably from 0 to 20 minutes, more preferably from 5 to 15 minutes.

 Specifically, ethanol may be added to the above supernatant obtained after centrifugation for sterilization, and the supernatant to which ethanol is added may be used as a ceramide production promoter and/or a glucosylceramide production promoter.

 The cells can also be removed from the culture solution by centrifugation, ultrasonically treated, centrifuged, and the supernatant is recovered. Next, the recovered supernatant is filtered to remove impurities and the like, and dried under vacuum to obtain a ceramide and/or glucosylceramide production-promoting agent of the present invention. The soybean residue fermentation extract obtained by culturing the yeast of the present invention and obtained from the culture supernatant has an effect of increasing the amount of ceramide and/or glucosylceramide in keratinocytes of normal humans.

 Therefore, the soybean residue fermentation extract obtained by cultivating the yeast of the present invention and obtained from the culture supernatant can be used for promoting therapeutic or non-therapeutic purposes of ceramide and/or glucosylceramide production, and can also be used as a treatment. Use of a ceramide production enhancer and/or a glucosylceramide production promoter for the purpose or non-therapeutic purposes. Use for non-therapeutic purposes is for cosmetic purposes, or for maintaining a healthy state of use. Moreover, the use of the therapeutic purpose and the use of non-therapeutic purposes can be performed in a completely differentiated manner.

 The soybean residue fermentation extract obtained by culturing the yeast of the present invention and obtained from the culture supernatant can also be used for producing a ceramide production promoter and/or a glucosylceramide production promoter.

The ceramide production promoting agent and/or the glucosylceramide production promoting agent of the present invention can be used as a pharmaceutical or quasi-medicine for increasing and recovering ceramide and/or glucosylceramide in the stratum corneum and improving the barrier function and moisturizing function of the skin. Use of products, cosmetics, etc. In addition, the ceramide production promoter or glucosylceramide production promoter can also be used as The use of quasi-drugs and cosmetics, which is based on the concept of ceramide production promotion or promotion of glucosylceramide production, and which indicates the concept as needed.

 The form of use of the ceramide production promoter and/or the glucosylceramide production promoter of the present invention may be an additive for addition to cultured cells, or may be incorporated into a skin external preparation such as a skin cleansing agent or a cosmetic. use. For example, various forms such as a lotion, an emulsion, a gel, a cream, an ointment, a powder, and a granule may be mentioned. The base as the external preparation is not particularly limited as long as it is a known external base. In the preparation of various skin external preparations, the ceramide production promoting agent and/or the glucosylceramide production promoting agent of the present invention or the oily component usually blended with the skin external preparations such as skin cleansing agents and cosmetics may be used singly. And a suitable combination of a moisturizer, a powder, a pigment, an emulsifier, a solubilizer, a UV absorber, a thickener, a medicinal ingredient, a fragrance, an antifungal agent, a plant extract, an alcohol, and the like.

 When the ceramide production promoter and/or the glucosylceramide production promoter of the present invention is added to cultured epidermal cells to promote ceramide and/or glucosylceramide, the amount of the dried product as the accelerator is preferably added. It is 0.0001 to 10 w/v%, more preferably 0.001 to 5 w/v%. When the addition amount is 0.0001 w/v% or more, sufficient promotion effect can be obtained; and when the addition amount is 10% or less, the irritation to epidermal cells is smaller.

 When the ceramide production promoter and/or the glucosylceramide production promoter of the present invention are used in an external preparation for skin, the production of ceramide and/or glucosylceramide is effectively promoted, thereby effectively improving the moisturizing ability of the skin, and is not easy The blending amount is preferably from 0.01 to 20 w/v%, more preferably from 0.1 to 10 w/v%, based on the total amount of the external preparation for skin, from the viewpoint of the color and odor of the culture. Example

 Hereinafter, the present invention will be described in more detail based on the examples, but the present invention is not limited thereto.

<Preparation of ceramide/glucosylceramide production promoter (fermentation liquid treatment) > First, the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. Broken, filter it The liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentrations of the okara and glucose in the mixture were respectively 10 w/v ° / ^ B lw / v%, and the mixture was used as a medium.

 Then, 100 ml of the above medium was added to an Erlenmeyer flask and inoculated with a certain amount of ISchizosaccharomyces octoporus (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0123), cultured at 30 °C. 3 days.

 The resulting culture was then centrifuged to GOOO rpm for 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and the diluted ethanol mixture was sonicated for 10 minutes, centrifuged at 80 rpm for 30 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper. The filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer, and the dried product was used as the sample 1 - A, 1 - B of the ceramide and/or glucosylceramide production promoter of the present invention, and the sample 1 - A, 1 was used. Each of -B was dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%, and stored in a refrigerator at 4 ° C as an additive for cell culture.

<Evaluation of ceramide/glucosylceramide production promoter>

(Cell culture) - First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at 37 ° C, C0 ₂ concentration. The cells were cultured to a concentration of 80 to 90% under conditions of 5%. After passage, the cells were passaged into a 175 cm ² square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per cell at a cell concentration of 1×10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced in the above 12-well plate, and samples 1 -A, 1 -B and a control (concentration of 5 (% by weight aqueous ethanol solution) were separately added to the above 12-well plates (n = 2), cultured for 3 days under the same culture conditions.

 After 3 days of culture, discard the supernatant medium, wash the cells twice with 2 ml of PBS (phosphate buffer), add 1 ml of PBS to the wells, and collect the cells into the tubes with a cell harvester (cell shovel). In order to prepare for subsequent ceramide/glucose ceramide and protein analysis.

(Confirmation of ceramide/glucose ceramide production promoting effect) Methanol: chloroform = 2.5 ml: 1.25 ml of a solvent was added to the recovered PBS-containing solution, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant a and a precipitate b. Supernatant a was used for the analysis of the amount of ceramide/glucosylceramide, and precipitate b was used for the analysis of protein content.

 (1) Analysis of ceramide/glucose ceramide amount

 To the supernatant a thus obtained, PBS: chloroform = 1.25 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain two upper and lower phases. The lower phase was recovered and dried under a nitrogen atmosphere for 30 minutes. To the obtained dried product, 100 / l of chloroform was added: methanol = 2 ml: 1 ml of a solvent was dissolved, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and GlyCer, the unit is ut g/ml).

 Place the ceramide (or glucosylceramide) standard and sample solution on a dry TLC plate, and perform thin-layer chromatography with CHCB: CH30H: CH3COOH (190: 9: 1 v/v/v) as a layering agent. The layer is applied to the top of the sheet and the spreader is blown dry with a blower; the above steps are repeated once. Then use CHCB: CH30H: C3H60 (76: 20: 4 v/v/v) as the layering agent for thin-layer chromatography, and wait for the layering agent to travel 2.5 cm from the bottom of the thin plate, stop the layer, and blow the hair with a hair dryer. . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatograph on a baking plate at 180 °C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucose ceramide).

 (2) Analysis of protein content

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. 90 μl of SDS (sodium dodecylsulfonate) solution was added to the above precipitate b and mixed, and the protein was denaturing and degraded by heating at 60 ° C for 2 hours, and after cooling, a 2N HCl solution of ΙΟΟμΙ was added thereto. The amount of protein (denoted as Pro, in g/ml) was determined according to the BCA Kit (Protein Quantitative Analysis Kit) method.

 (3) Analysis results

For the samples of the present invention and the control, Cer/Pro and GlyCer Pro (see the following formula) were respectively determined. The relative values of Cer/Pro and GlyCer/Pro of the present invention are shown in Table 1 with the Ser/Pro and GlyCer Pro of the control being 1. Further, the relative amount (Pro. (%)) of the amount of protein in each well of the present invention was determined by taking the amount of protein in each well of the control product as 100, and the ceramide and each well were determined accordingly. Amount of glucosylceramide (relative to the control) The relative amount), that is, Cer/Well=Cer/Pro X Pro. (%), GlyCer/Well=GlyCer/Pro X Pro. (%)c is shown in Table 1.

 If Cer Pro (or Cer/Well) is greater than 1 (the value of the control), this sample has a ceramide production promoting effect; if GlyCer/Pro (or GlyCer/Well) is greater than 1 (the value of the control), it indicates This sample has a glucose ceramide production promoting effect.

 As shown in Table 1, it was confirmed that the inventive products 1-A and 1-B have a ceramide production promoting effect and/or a glucosylceramide production promoting effect. Table 1

另外， 对于样品 1-A〜1-B, 不将其加入到上述含角质化细胞的 12 孔板中 （即， 不进行上述细胞培养） 而直接进行神经酰胺 /葡萄糖神经 酰胺量的分析，除此以外的操作与上述实施例 1-1同样进行。结果确认 了样品 1-A〜1-B本身中没有神经酰胺或葡萄糖神经酰胺存在。

Further, for the samples 1-A to 1-B, the ceramide/glucose ceramide amount was directly analyzed without adding it to the above-mentioned 12-well plate containing keratinocytes (that is, without performing the above cell culture). The other operations were carried out in the same manner as in the above Example 1-1. As a result, it was confirmed that no ceramide or glucosylceramide was present in the samples 1-A to 1-B themselves.

 This indicates that ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent. Example 2-1

<Preparation of ceramide/glucosylceramide production promoter (fermentation liquid treatment) > First, the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. It is crushed, filtered and the liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentrations of the okara and glucose in the mixture were respectively 10 w/ 3⁄4^n lw/v%, and the mixture was used as a medium. Then, 100 ml of the above medium was added to the Erlenmeyer flask and inoculated with a certain amount of Pichia anthracis (Pichia an awakening (preserved at the Industrial Microbial Resources and Information Center of Jiangnan University, CICIM, No. 360-20-3) at 30°. C culture for 3 days.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucosylceramide-producing agent-producing sample 2-A of the present invention, and stored in a refrigerator at 4 ° C for use in cell culture. Additives.

<Evaluation of ceramide/glucosylceramide production promoter>

 (cell culture)

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at 37 ° C and a CO ₂ concentration of 5%. The cells were cultured to a concentration of 80 90%. After passage, the cells were passaged into a 175 cm ² square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per well, and the cell concentration was IX 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced in the above 12-well plate, and the sample 2-A and the control (concentration of 5 (% by weight aqueous ethanol solution) were added to the above 12-well plate (n=2), and the same culture was carried out. Incubate for 3 days under the conditions.

 After 3 days of culture, discard the supernatant medium, wash the cells twice with 2 ml of PBS (phosphate buffer), add 1 ml of PBS to the wells, and collect the cells into the tubes with a cell harvester (cell shovel). In order to prepare for subsequent ceramide/glucose ceramide and protein analysis.

 (Confirmation of ceramide/glucose ceramide production promoting effect)

 Add methanol to the recovered cell-containing PBS solution: chloroform = 2.5 ml:

1.25 ml of the solvent was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to separate, thereby obtaining supernatant a and precipitate b. The supernatant a was used for the analysis of the amount of ceramide/glucose amide, and the precipitate b was used for the analysis of the protein content.

 (1) Analysis of ceramide/glucose ceramide amount

To the thus obtained supernatant a, PBS: chloroform = 1.25 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged for 5 minutes to separate the upper and lower phases. The lower phase was recovered and dried under a nitrogen atmosphere of 30 C for 40 minutes. To the obtained dried product, chloroform of ΙΟΟ μ Ι was added: methanol = 2 ml: 1 ml of a solvent was dissolved, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and Analysis of GlyCer, in μg/ml).

 The ceramide (or glucosylceramide) standard and sample solution are spotted onto a dry TLC plate and chromatographed with CHC13 : CH30H: CH3COOH (190: 9: 1 v/v/v) as a layering agent. The layer is applied to the top of the sheet, and the layer spreader is blown by a blower; the above steps are repeated once. Then use CHCB: CH30H: C3H60 (76: 20: 4 v/v/v) as the layering agent for thin-layer chromatography, and wait for the layering agent to travel 2.5 cm from the bottom of the sheet, stop the layer, and blow dry with a hair dryer. . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatograph on a baking plate at 180 °C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucose ceramide).

 (2) Analysis of protein content

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. 90 μl of SDS (sodium dodecylsulfonate) solution was added to the above precipitate b and mixed, and the protein was denaturing and degraded by heating at 60 ° C for 2 hours, and after cooling, 2 μ of HCl solution of ΙΟΟμΙ was added thereto. The amount of protein (denoted as Pro, in g/ml) was determined according to the BCA Kit (Protein Quantitative Analysis Kit) method.

 (3) Analysis results '

For the samples of the present invention and the control, Cer/Pro and Gl _y Cer/Pro were respectively determined (see the following formula). The relative values of Cer/Pro and GlyCer/Pro of the present invention are shown in Table 2 as Ser/Pro and GlyCer/Pro of the control. In addition, the relative value (Pro. (%)) of the amount of protein in each well of the present invention was determined by taking the amount of protein in each well of the reference product as 100, and the nerve of each well was determined accordingly. The amount of amide and glucosylceramide (relative to the relative amount of the control), SP, Cer/Well=Cer/Pro X Pro. (%), GlyCer/Well=GlyCer/Pro X Pro. (%) o Table 2 shows.

 If Cer/Pro (or Cer/Wdl) is greater than 1 (the value of the control), this sample has a ceramide production-promoting effect; if GlyCer/Pro (or GlyCer/Well) is greater than 1 (the value of the reference), then This sample was shown to have a glucose ceramide production promoting effect.

As shown in Table 2, it was confirmed that the product 2-A of the present invention has a ceramide production promoting effect and a glucosylceramide production promoting effect. Example 2-2

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondiO (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, No. 422-19-3) Instead of the abnormal Pichia anomala, and diluted with 2.5 times of absolute ethanol in the supernatant obtained after the fermentation of the okara, the mixture was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant was collected. 70 ml), the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucosylceramide production promoter of the present invention 2-B, 2-C, and the dried sample was 50. The % ethanol was dissolved, and the samples 2-B and 2-C were each prepared into a 1%, 0.1% concentration solution, and stored in a refrigerator at 4 ° C as an additive for cell culture. Except for the same procedure as in Example 2-1, the ceramide/glucosylceramide production-promoting effect of Samples 2-B and 2-C was evaluated, and the results are shown in Table 2.

 As shown in Table 2, it was confirmed that the inventive products 2-B and 2-C have a ceramide production promoting effect. Example 2-3

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia norvegensis (CICIM, numbered CICIM Y015 1 deposited at the Jiangnan University Industrial Microbial Resources and Information Center) was used instead of the abnormality. In the same manner as in Example 2-1, except that Pichia anomala was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the sample 2-D of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use in cell culture. Additives.

The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. Samples 2-E and 2-F, which are ceramide/glucosylceramide production promoters of the present invention, were prepared by dissolving the samples 2-E and 2-F in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture. The ceramide/glucose ceramide production promoting effect of the samples 2-D, 2-E, and 2-F was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2.

 As shown in Table 2, it was confirmed that the samples 2-D, 2-E, and 2-F of Example 2-3 had a ceramide-promoting effect. Example 2-4

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the Pichia pastoris (a Pichi discussion! a (preserved at the Industrial Microbial Resources and Information Center of Jiangnan University, CICIM, No. CICIM Y0297) was used instead. The culture was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 2-G of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged

GOOOrpm ) 10 minutes, the supernatant (about 70 ml) was collected, the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight as a sample of the ceramide/glucose ceramide-producing agent of the present invention. 2-H, the sample 2-H was dissolved in 50% ethanol to prepare a 1% strength solution, and stored in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucose ceramide production promoting effect of the samples 2-G and 2-H was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

 As shown in Table 2, it was confirmed that the samples 2-G and 2-H of Example 2-4 have a ceramide and glucosylceramide production-promoting effect. Example 2-5

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the anomalous Pichia was used to replace mala (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0349). In the same manner as in Example 2-1, except that the yeast Pichia anomala) (No. 360-20-3) was used.

The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and 1 ml of the mixture was taken as the ceramide of the present invention/ Sample 2-1 of the glucosylceramide production promoter was stored in a refrigerator at 4 ° C for use as an additive for cell culture.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. As a sample of the ceramide/glucosylceramide production promoter of the present invention, 2-J and 2-K, the samples 2-J and 2-K were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. Stored in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucose ceramide production promoting effect of the samples 2-1, 2-J, and 2-K was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

 As shown in Table 2, it was confirmed that the samples 2-1, 2-J, and 2-K of Example 2-5 had a ceramide and/or glucosylceramide production-promoting effect. Example 2-6

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia membranifaciens ( deposited in the Industrial Microbial Resources and Information Center of Cangnam University, number CICIM Y0360) was used instead of the abnormality. The same was carried out in the same manner as in Example 2-1 except that the yeast (i3⁄4/^ "oma/) (No. 360-20-3) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucose ceramide production promoter sample 2-L of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. Samples 2_M and 2-N, which are ceramide/glucosylceramide production promoters of the present invention, were prepared by dissolving the samples 2-M and 2-N in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was used as an additive for cell culture in a refrigerator at 4 °C.

The ceramide/glucose ceramide production promoting effect of the samples 2-L, 2-M, and 2-N was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together. As shown in Table 2, it was confirmed that the samples 2-L, 2-M, and 2-N of Example 2-6 had a ceramide production promoting effect. Example 2-7

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the Pichia pastoris iPichia cmomala (preserved at the Industrial Microbial Resources and Information Center of Jiangnan University, CICIM Y0420) was substituted for P. angustifolia. In the same manner as in Example 2-1, except that Pichia anomala) (No. 360-20-3) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the sample 2-0 of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. Samples 2-P and 2-Q, which are ceramide/glucosylceramide production promoters of the present invention, were prepared by dissolving the samples 2-P and 2-Q in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. , stored in a refrigerator at 4 ° C as an additive for cell culture

 The ceramide/glucose ceramide production promoting effect of the samples 2-0, 2-P, and 2-Q was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

 As shown in Table 2, it was confirmed that the samples 2-0, 2-P, and 2-Q of Example 2-7 have a ceramide-promoting-promoting effect. Example 2-8

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia anomala (CICIM, No. CICIM Y0421, deposited in the Industrial Microbial Resources and Information Center of Jiangnan University) was replaced by Pichia anomala In the same manner as in Example 2-1, except that ί lochia anomala) (No. 360-20-3) was used.

The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and 1 ml of the mixture was taken as the ceramide of the present invention/ Sample 2-R of the glucosylceramide production promoter was stored in a refrigerator at 4 ° C for use as an additive for cell culture.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. As a sample of the ceramide/glucosylceramide production promoter of the present invention, 2-S, 2-T, the samples 2-S and 2-T were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. Stored in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucose ceramide production promoting effect of the samples 2-R, 2-S, and 2-T was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

 As shown in Table 2, it was confirmed that the samples 2-R, 2-S, and 2-T of Example 2-8 had a ceramide-promoting effect. Example 2-9

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia burtonii (CICIM, No. CICIM Y0424, deposited at the Industrial Microbial Resources and Information Center of Jiangnan University) was replaced by Abnormal Pichia. The yeast (No. 360-20-3) was cultured in the same manner as in Example 2-1.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the sample 2-U of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. As a sample 2-V of the ceramide/glucosylceramide production promoter of the present invention, the sample 2-V was dissolved in 50% ethanol to prepare a 1% concentration solution, and stored in a refrigerator at 4 ° C for cell culture. Additives.

 The ceramide/glucosylceramide production promoting effect of the samples 2-U and 2-V was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

As shown in Table 2, it was confirmed that the samples 2-U and 2-V of Example 2-9 have a ceramide production promoting effect. Example 2 - 10

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondii (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0440) was replaced. The culture was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucose ceramide production promoter sample 2-W of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The ceramide/glucose amide production promoting effect of the sample 2-W was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

 As shown in Table 2, it was confirmed that the sample 2-W of Example 2-10 had a glucosylceramide production promoting effect. Example 2 - 1 1

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondi (contained at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0326) was used instead of the abnormality. The same was carried out in the same manner as in Example 2-1 except that the yeast (No. 360-20-3) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 2-X of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged

OOOOrpm) 10 minutes, the supernatant (about 70 ml) was collected, the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucose ceramide production promoter of the present invention. -Y, the sample 2-Υ was dissolved in 50% ethanol to prepare a 0.1% strength solution, and stored in a refrigerator at 4 ° C as an additive for cell culture.

The ceramide/glucose ceramide production promoting effect of the sample 2-X, 2-Y was evaluated in the same manner as in Example 2-1, and the results are shown together in Table 2. As shown in Table 2, it was confirmed that the samples 2-X and 2-Y of Example 2-11 have a glucosylceramide production-promoting effect. Example 2 - 12

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the Pichia guilliermondii) (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0329) was used instead of the abnormality. The same was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 2-Z of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged (3000 rpm) for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant temperature. As a sample 2-a of the ceramide/glucose ceramide production promoter of the present invention, the sample 2-a was dissolved in 50% ethanol to prepare a 1% concentration solution, and stored in a refrigerator at 4 ° C as a cell culture. Use additives.

 The ceramide/glucosylceramide production promoting effect of the samples 2-Z and 2-a was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.

 As shown in Table 2, it was confirmed that the samples of Examples 2-12 2-Z, 2-a have ceramide and

/ or glucosyl ceramide production promoting effect. Example 2- 13

 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondii (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, No. CICIM Y0414) was replaced. The culture was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.

The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper. The filtrate is obtained, and the filtrate is dried in a vacuum dryer to a constant weight as a Sample 2-b of the ceramide/glucosylceramide production promoter of the invention was prepared by dissolving the sample 2-b in 50% ethanol to prepare a 1% strength solution, and storing it in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucose amide production promoting effect of the sample 2-b was evaluated in the same manner as in Example 2-1, and the results are shown together in Table 2.

 As shown in Table 2, it was confirmed that the sample 2-b of Example 2-13 had a ceramide and glucose ceramide production promoting effect. Table 2

 Guilliermondii) In addition, for samples 2-A~2-Z and 2-a, 2-b, they are not added to the above-mentioned 12-well plates containing keratinocytes (ie, without the above cell culture) The analysis of the amount of ceramide/glucosylceramide was carried out, and the other operations were carried out in the same manner as in the above Example 2-1. As a result, it was confirmed that there were no ceramide or glucosylceramide present in the samples 2-A to 2-Z and 2-a, 2-b themselves.

This indicates that ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent. Example 3-1 (Manufacture of ceramide and/or glucosylceramide production promoter)

 As a microorganism for producing ceramide and/or glucosylceramide production promoter from bean dregs, Candida zeylanoides (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 472) -20-1).

 The above-mentioned strain obtained by culturing in YPD (Yeast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into a conical flask (capacity 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours. The medium contained 10 w/v% okara and lw/v% glucose. Among them, the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.

 Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. After dilution with 2.5 times of absolute ethanol, 1 ml was taken as a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 3-Α-1).

 Then, the crude product was treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifuged at 3000 rpm for 30 minutes, and the supernatant was recovered. The supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract is dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract is adjusted to be 1 w/v% (inventive product 3-A-2), and then stored at 4 ° C for use in the subsequent Ceramide/glucose ceramide production promotion test. Example 3-2

As a microorganism for producing ceramide/glucose ceramide production promoter from bean dregs, Candida zeylanoides was purchased from Jiangnan University and preserved at CICIM, China University Industrial Microbial Resources and Information Center, original number 406-20 -1 ). The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention 3-B-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product. As the ceramide/glucosylceramide production promoter of the present invention. Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-B-2) 0.1 w/v% (inventive product 3-B-3). Stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion assays. Example 3-3

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida parapsilosis was used (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 5 14- 20-3). The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention 3-C-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product. As the ceramide/glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (%% ethanol), and the concentration thereof was adjusted to 1 w/v% (the present invention 3-C-2), and then stored at 4 ° C for the subsequent ceramide/glucose. Ceramide production promotion test. Examples 3-4

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Ccmdi parapsilosis (Ccmdi parapsilosis) (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 525- 20-2). The soybean residue fermentation extract was obtained in the same manner as in Example 3-1.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to 1 w/v% (3-D1 of the present invention) and 0.1 w/v% (3-D-2 of the present invention). Stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion assays. Examples 3-5

As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida metapsilosis is used (purchased from Jiangnan University and preserved at CICIM, China University Industrial Microbial Resources and Information Center, original number 510-19 -3 ). The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention 3-E-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product. As the ceramide/glucosylceramide production promoter of the present invention. Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-E-2) and 0.1 w/v% (inventive product 3-E-3). Thereafter, it was stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion test. Example 3-6

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida parapsilosis (purchased from Jiangnan University and deposited in CICIM, China University Industrial Microbial Resources and Information Center, under the accession number CICIM Y0314) o The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (3-F-1 of the present invention) was obtained in the same manner as in Example 3-1, and the crude soybean obtained by the crude product was fermented and extracted. The ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (the present invention 3-F-2) and 0.1 w/v% (the present invention 3-F-3). Thereafter, it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test. Example 3-7

 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Candida parapsilosis ( purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0322 ). In the same manner as in Example 3-1, a crude product of the ceramide and/or glucose ceramide production promoter of the present invention (the present invention 3-G-1) was obtained, and the crude product was further purified by the crude product. As a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-G-2), 0.1 w/v% (inventive product 3-G-3). Thereafter, it was stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion test. Example 3-8

As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida boidini (candida boidini (purchased from Jiangnan University and preserved in China University Industrial Microbial Resources and Information Center CICIM, Accession No. CICIM Y0366 The crude product of the ceramide and/or glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1 (Inventive product 3-H-1) Further, the soybean residue fermentation extract obtained by purifying the crude product is used as the ceramide/glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to 1 w/v% (the present invention, 3-H-2), and then stored at 4 Torr for use in the subsequent ceramide/glucosylceramide. A boost test was generated. Examples 3-9

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida metapsilosis (purchased from Jiangnan University and preserved in CICIM, China University Industrial Microbial Resources and Information Center, with the accession number CICIM Y0439) The soybean residue fermentation extract was obtained in the same manner as in Example 3-1.

 Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-1-1) and 0.1 w/v% (inventive product 3-1-2). Stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion assays. Example 3-10

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida parapsilosis (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number C1CIM Y0443) The soybean residue fermentation extract was obtained in the same manner as in Example 3-1.

Further, use 50 _V /v ° /. Ethanol dissolves the soybean residue fermentation extract, and adjusts the concentration to lw/v% (inventive product 3-Jl) and 0.1 w/v% (inventive product 3-J-2), respectively, and stores at 4 ° C. For subsequent ceramide/glucose ceramide production promotion experiments. Example 3-11

 As a microorganism for producing ceramide/glucosylceramide production promoter from bean dregs, Candida utilis Ccmdidci magnoli ^ (purchased from Jiangnan University and preserved in

】 China University Industrial Microbial Resources and Information Center CICIM, Accession No. CICIM Y0184) o The soybean residue fermentation extract was obtained in the same manner as in Example 3-1.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-Κ-1), 0.1 w/v% (inventive product 3-K-2). Thereafter, it was stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion test. Example 3-12

 As a microorganism for producing ceramide/glucose ceramide production promoter from bean dregs, Candida maltoscO (Candida maltoscO (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0207) o The soybean residue fermentation extract was obtained in the same manner as in Example 3-1.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration was adjusted to 0.1 w/v% (3-L-1 of the present invention), and then stored at 4 ° C for the subsequent ceramide/ Glucose ceramide production promotion test. Example 3-13

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida lipolytica (Owi3⁄43⁄4 W^to/_y/c) (purchased from Jiangnan University and deposited in the Industrial Microbial Resources and Information Center of China University of China) CICIM, deposit number CICIM Y0221). In the same manner as in Example 3-1, a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (3-Μ-1 of the present invention) was obtained, and the crude soybean product fermentation extract was obtained by further purifying the crude product. As a ceramide/glucose ceramide production promoter of the present invention, it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test. Example 3-14

As a microorganism for producing ceramide/glucose ceramide production promoter from bean dregs, Candida butyricum (Ccmdkia imtyrO (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0233) The soybean residue fermentation extract was obtained in the same manner as in Example 3-1. Further, the soybean residue fermentation extract was dissolved in 5 (%% ethanol), and the concentration was adjusted to 1 w/v% (the present invention 3-N-1), and then stored at 4 ° C for the subsequent ceramide/ Glucosylceramide production promotion test. Examples 3-15

 As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Candida rhagi O (Candida rhagi O (purchased from Jiangnan University and deposited at the China University Industrial Microbial Resources and Information Center CICIM, accession number CICIMY0248) was used. In the same manner as in Example 3-1, a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 3-0-1) was obtained, and the crude soybean product fermentation extract was further purified. As the ceramide/glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-0-2) and 0.1 w/v% (inventive product 3-0-3). Thereafter, it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test. Example 3-16

 As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Candidazeylanoide (purchased from Jiangnan University and preserved at the China University Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0428) was used. The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention 3-P-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product. As the ceramide/glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 50 ν / ν% ethanol, adjusted to a concentration of lw / v% (3-P-2 of the present invention), and stored at 4 ° C for subsequent ceramide / glucose Ceramide production promotion test. Example 3-17

As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, use Candida parapsilosis (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM) Y0325). The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention, 3-Q-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product. As the ceramide/glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (%% ethanol), and the concentration was adjusted to be lw/v%.

(Inventive product 3-Q-2) was stored at 4 ° C for subsequent ceramide/glucose amide production promotion test.

(ceramide/glucose ceramide production promotion test)

 <Keratinocytes culture>

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at a concentration of 5% at 37 ° C and C0 ₂ . The cell culture was carried out under the conditions until the cell converence was 80 to 90%. Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at IX 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced, and the aqueous solution of the soybean residue fermentation extract obtained in the above Examples 3-1 to 3-17 was added to the above-mentioned 12-well plate (n=2) in an amount of 20 μl/well. . Further, only 50 Wv% of ethanol (without addition of a ceramide/glucosylceramide production promoter) was added as a control, and culture was carried out for 72 hours under the same culture conditions. After 72 hours of culture, discard the supernatant culture solution.

 The cells were washed twice with 2 ml PBS (phosphate buffer), and 1 ml of PBS was added to the wells, and the cells were collected into a test tube by a cell harvester (cell shovel) for subsequent ceramide/glucose ceramide and protein analysis.

<Lipid extraction>

Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, methanol was added to the recovered cell-containing PBS solution: chloroform = 2.5 ml: 1.25 ml of a solvent, and the mixture was shaken for 20 minutes, and centrifuged to separate GOOO rpm for 5 minutes) to obtain a supernatant and precipitation. Supernatant A was transferred to an additional tube for analysis of ceramide/glucose ceramide amount and Precipitate B was used for analysis of protein content. <Protein Content Analysis>

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. Add 900 μl of SDS (sodium dodecyl sulfate) solution to the precipitate B recovered above, mix it, and heat-degrade the protein for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, add 2N HCl solution of ΙΟΟμΙ to it. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Kit).

<Ceramid/Glucosylceramide Amount Analysis>

 To the remaining supernatant A, PBS: chloroform = 1.25 mh 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, centrifuged at 3000 rpm for 5 minutes, and the chloroform layer as a lower phase was separated and recovered, and protected at 30 ° C with nitrogen gas. The dried chloroform layer was dried for 40 minutes to obtain a dried product, which was dissolved in chloroform: methanol = 2.5 ml: 1.25 ml of a solvent, and the amount of ceramide and glucosylceramide was determined by the following thin plate chromatography (TLC) method (respectively Cer and GlyCer, in g/ml) were quantified.

 Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9:1 v/v/v) as a layering agent for thin-layer chromatography, the layering agent is applied to the top of the sheet, and the layering agent is blown dry with a hair dryer; The above steps are once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate on a baking plate at 180 ° C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucosyl ceramide). '

 Then, according to the following formula (1), the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The relative values of Cer/Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 3.

 Formula 1 )

 Cer/Pro=Ceramic amide amount (g/ml) / protein content g/ml)

GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content ^g/ml) table 3

如表 3所示， 实施例 3-1、 实施例 3-6〜3-7以及实施例 3-17中得 到的本发明品既具有神经酰胺产生促进效果， 又具有葡萄糖神经酰胺 产生促进效果， 另外， 实施例 3-2、 实施例 3-9〜3-13以及实施例 3- 15 中得到的本发明品的葡萄糖神经酰胺产生促进效果显著， 而实施例 ₃_3〜3-5、 实施例 3-8、 实施例 3-14以及实施例 3-16中得到的本发明 品的神经酰胺产生促进效果显著。

As shown in Table 3, the inventive product obtained in Example 3-1, Examples 3-6 to 3-7, and Example 3-17 had both a ceramide production promoting effect and a glucosylceramide. In addition, the glucosylceramide production-promoting effect of the present invention obtained in Example 3-2, Examples 3-9 to 3-13, and Example 3-15 was remarkable, and Example ₃ _3 to 3- 5. The ceramide production promoting effect of the present invention obtained in Examples 3-8, Examples 3-14 and Examples 3-16 was remarkable.

 Therefore, it has been confirmed that the ceramide/glucose ceramide production promoting agent of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.

(Test for the absence of ceramide and glucosylceramide in the ceramide/glucosylceramide production promoter of the present invention)

 The ceramide/glucosylceramide production promoter obtained in the above Examples 3-1 to 3-17 was directly analyzed by TLC without cell culture, and as a result, no ceramide and glucosylceramide were present in the present invention.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or a glucose ceramide production promoter. use. Example 4- 1

 (Manufacture of ceramide and/or glucosylceramide production promoter)

 As a microorganism producing ceramide and/or glucosylceramide-producing agent from bean dregs, Debaryomyces hansenii was used (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 476 -20-1).

The above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours. The medium contained 10 w/v% okara and 1 w/ _v % glucose. Among them, the bean dregs are prepared by soaking the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.

Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the cells are recovered after centrifugation. The supernatant was added to the culture supernatant after the recovery, and 2.5 times of absolute ethanol was added thereto, and the mixture was pulverized by an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifuged at 3000 rpm for 30 minutes, and the supernatant was recovered. The supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to be lw/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. Example 4-2

 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, Debatyomyces hansenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 5) 14-20-4). The soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. Example 4-3

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Debaryomyces hansenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM) Y0356) o The soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

Further, the soybean residue fermentation extract was dissolved in 5 (% ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. Example 4-4 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Debaryomyces hansenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM) Y0286 A soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 C for subsequent ceramide/glucose ceramide production promotion test. 4-5

 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Debaryomyces hansenii) (purchased from Jiangnan University and preserved in CICIM, China University Industrial Microbial Resources and Information Center, under the accession number iciM Y0287 The soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5% ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. Example 4-6

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Debaryomyces hansenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM) Y0289 A soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for use in the subsequent ceramide/glucose ceramide production promotion test. Example 4-7 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Debaryomyces hansenii) (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0290 ). The soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to be lw/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. Example 4-8

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, use B. vannamei ebaryomyces vanrijiae (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0198 adopted and implemented Example 4 - 1 The same method was used to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 0.1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. Example 4-9

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Debaiyomyces hansenii (purchased from Jiangnan University and deposited in CICIM, China University Industrial Microbial Resources and Information Center, CICIM) Y035 1 A soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5% ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. (ceramide/glucose ceramide production promotion test)

<Keratinocytes culture> First, activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium), and cells were cultured at 37 ° C and a CO ₂ concentration of 5%. The culture is maintained until the cell aggregation density (concentration) is 80 to 90%. Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at 1 × 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced, and the lw/v% bean dregs fermentation extracts of the above Examples 4-1 to 4-9 were added in the above 12-well plates (n=2) in an amount of 20 μl/well. An aqueous solution of ethanol. In addition, only 50 v/v% ethanol (without addition of ceramide/glucosylceramide production promoter) was added as a control, and cultured under the same culture conditions for 72 hours. After 72 hours of culture, the supernatant culture solution was discarded, and the cells were washed twice with 2 ml of PBS (phosphate buffer) (2 ml/well), and then 1 ml of PBS was added to the wells, and the cells were collected by a cell harvester (cell shovel). Into the test tube for subsequent ceramide/glucose ceramide and protein analysis.

<Lipid extraction>

 Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, a solvent of methanol: chloroform = 2.5 mh 1.25 ml was added to the recovered cell-containing PBS solution, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant and a precipitate. . Supernatant A was transferred to another tube for analysis of ceramide/glucose ceramide amount, and precipitate B was used for analysis of protein content.

<Protein Content Analysis>

To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. 900 μl of SDS (sodium dodecyl sulfate) solution was added to the precipitate B recovered above, and mixed, and heated and dehydrated for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, a 2N HC solution of ΙΟΟμΙ was added thereto. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Assay Kit) method. <Ceramid/Glucosylceramide Amount Analysis> Add PBS: chloroform = 1.25 ml: 1.25 ml of solvent to the remaining supernatant A, shake for 20 minutes, centrifuge at 3000 rpm for 5 minutes, separate and recover the chloroform layer as the lower phase, and protect it at 3 (TC, nitrogen) The chloroform layer was dried for 40 minutes to obtain a dried product, and chloroform: methanol = 2.5 ml: 1.25 ml of a solvent was added, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and GlyCe units are quantified in g/ml).

 Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9:1 v/v/v) as a layering agent for thin-layer chromatography, the layering agent is applied to the top of the sheet, and the layering agent is blown dry with a hair dryer; The above steps are once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate on a baking plate at 180 ° C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucosyl ceramide).

 Then, according to the following formula (1), the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The relative values of Cer Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 4.

 Formula 1 )

 Cer/Pro=Ceramic amide amount g/ml) /protein content ( g/ml )

 GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content (g/ml)

4

 Cer Pro Cer/Well GlyCer/Pro GlyCer/Well

Reference 1 1 1 1

Example 4-1 2.68 3.17 2.29 2.70

Example 4-2 2.57 3.02 0.61 0.71

Example 4-3 0.09 0.12 1.32 1.66

Example 4-4 3.77 4.99 0.36 0.48

Example 4-5 3.05 3.71 0.20 0.25

Example 4-6 1.55 1.46 0.34 0.32

Example 4-7 1.03 1.38 0.07 0.10

Example 4-8 0.99 0.91 1.13 1.04 Example 4-9 1.53 1.40 1.35 1.23 As shown in Table 4, the inventive product obtained in Example 4-1 and Example 4-9 has both a ceramide production promoting effect and a glucosylceramide production promoting effect; The ceramide production-promoting effect of the present invention obtained in Example 4-2 and Examples 4-4 to 4-7 was remarkable, and the glucose of the present invention obtained in Example 4-3 and Example 4-8 was obtained. The ceramide production promoting effect is remarkable. Therefore, it was confirmed that the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect. (Test for the absence of ceramide and glucosylceramide in the ceramide/glucosylceramide production promoter of the present invention)

 The ceramide/glucosylceramide production promoter obtained in the above Examples 4- 1 to 4-9 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide and glucosylceramide present in the present invention.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. .

<Preparation of ceramide/glucosylceramide production promoter (fermentation liquid treatment) > First, the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. It is crushed, filtered and the liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentration of the okara and glucose in the mixture was respectively 10 w/ _V . /^Q lw/v% , the mixture was used as a medium. '

 Then, 100 ml of the above medium was added to an Erlenmeyer flask, and a certain amount of Cryptococcus laurentii (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, No. CICIM Y0232) was inoculated and cultured at 30 °C. day.

The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and 1 ml of the mixture was taken as the ceramide of the present invention. Sample 5-A of the glucosylceramide production promoter was stored in a 4 C refrigerator for use as an additive for cell culture.

 The diluted ethanol solution was sonicated for 10 minutes, centrifuged at OOOO rpm for 30 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. The dried product was used as the sample 5-B, 5-C of the ceramide and/or glucosylceramide production promoter of the present invention, and the samples 5-B and 5-C were each dissolved in 50% ethanol to prepare 1%. A 0.1% strength solution was stored in a refrigerator at 4 ° C as an additive for cell culture. <Evaluation of ceramide/glucosylceramide production promoter>

 (cell culture)

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at 37 ° C and a CO ₂ concentration of 5%. The cells were cultured to a concentration of 80 to 90%. After passage, the cells were passaged into a 175 cm ² square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per well, and the cell concentration was IX 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced in the above 12-well plate, and samples 5-A, 5-B, 5-C and a control (concentration of 5 (% by weight aqueous ethanol solution) were separately added to the above 12-well plate. Medium (n=2), cultured for 3 days under the same culture conditions.

 After 3 days of culture, discard the supernatant medium, wash the cells twice with 2 ml of PBS (phosphate buffer), add 1 ml of PBS to the wells, and collect the cells into the tubes with a cell harvester (cell shovel). In order to prepare for subsequent ceramide/glucose ceramide and protein analysis.

 (Confirmation of ceramide/glucose ceramide production promoting effect)

 To the recovered cell-containing PBS solution, methanol: chloroform = 2.5 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant a and a precipitate b. The supernatant a was used for the analysis of the amount of ceramide/glucose amide, and the precipitate b was used for the analysis of the protein content.

(1) Analysis of ceramide/glucose ceramide amount To the supernatant a thus obtained, PBS: chloroform = 1.25 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain two upper and lower phases. The lower phase was recovered and dried under nitrogen at 40 ° C for 40 minutes. To the obtained dried product, 100 / il of chloroform was added: methanol = 2 ml: 1 ml of a solvent was dissolved, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and GlyCer, in units of / g/ml).

 The ceramide (or glucosylceramide) standard and sample solution are spotted onto a dry TLC plate and chromatographed with CHC13 : CH30H: CH3COOH (190: 9: 1 v/v/v) as a layering agent. The layer is applied to the top of the sheet and the spreader is blown dry with a blower; the above steps are repeated once. Then use CHCB: CH30H: C3H60 (76: 20: 4 v/v/v) as a layering agent for thin-layer chromatography, and wait for the layering agent to travel 2.5 cm from the bottom of the sheet, stop the layer, and blow dry with a hair dryer. . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatograph on a baking plate at 180 °C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucose ceramide).

 (2) Analysis of protein content

To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. 90 μl of SDS (sodium dodecylsulfonate) solution was added to the above precipitate b and mixed, and the protein was denaturing and degraded by heating at 60 ° C for 2 hours, and after cooling, 2 μ of HCl solution of ΙΟΟμΙ was added thereto. The amount of protein (denoted as Pro, in units of _μβ / ηι1) was determined according to the BCA Kit (Protein Quantitative Analysis Kit) method.

 (3) Analysis results

For the samples of the present invention and the control, Cer/Pro and GlyCer/Pro were respectively determined (see the following formula). The relative values of Cer/Pro and GlyCer/Pro of the present invention are shown in Table 5 with the Ser/Pro and GlyCer/Pro of the control being 1. In addition, the relative value (Pm. (%)) of the amount of protein in each well of the present invention was determined by taking the amount of protein in each well of the reference product as 100, and the nerve of each well was determined accordingly. The amount of amide and glucosylceramide (relative to the relative amount of the control), ie, Cer/Well=Cer/Pro X Pro. (%), GlyCer/Well=GlyCer/Pro X Pro. (%) ₀ Table 5 shows.

If Cer/Pro (or Cer/Well) is greater than 1 (the value of the control), this sample has a ceramide production promoting effect; if GlyCer/Pro (or GlyCer/Well) is greater than 1 (the value of the reference), then This sample was shown to have a glucose ceramide production promoting effect. As shown in Table 5, it was confirmed that the inventive products 5-A, 5-B and 5-C have an amide production promoting effect. Example 5-2

 In the preparation of the ceramide/glucose ceramide production promoter of Example 5-1, the Cryptococcus hungariciis (CICIM deposited in the Industrial Microbial Resources and Information Center of Jiangnan University, number CICIM Y0203) was used instead of the Loren Hidden Ball. The culture was carried out in the same manner as in Example 5-1 except for the yeast Cryptococc laurentii) (No. CICIM Y0232).

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as a sample 5-D of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. Samples 5-E and 5-F which are ceramide/glucosylceramide production promoters of the present invention, and samples 5-E and 5-F were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture.

 Samples 5-D, 5-E, 5-F ceramide were evaluated in the same manner as in Example 5-1.

/ Glucosylceramide production promoting effect, and the results are shown together in Table 5.

 As shown in Table 5, it was confirmed that the samples 5-D, 5-E, and 5-F of Example 5-2 have a glucose ceramide production promoting effect. Example 5-3

In the preparation of the ceramide/glucose ceramide production promoter of Example 5-1, the C. cerevisiae i Cryptococcus laurentii (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0218) was used instead of The culture was carried out in the same manner as in Example 5-1 except that Cryptococcus laurentii (No. CICIM Y0232) was used. The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 5-G of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged

(3000 rpm) 10 minutes, the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight as a sample of the ceramide/glucosylceramide production promoter of the present invention. 5-H, Sample 5-H was dissolved in 50% ethanol to prepare a 1% strength solution, and stored in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucosylceramide production promoting effect of the samples 5-G and 5-H was evaluated in the same manner as in Example 5-1, and the results are shown in Table 5.

 As shown in Table 5, it was confirmed that the samples 5-G and 5-H of Example 5-3 had a glucose ceramide production promoting effect. Example 5-4

 In the preparation of the ceramide/glucose ceramide production promoter of Example 5-1, the Cryptococcus hungaricus (CICIM, numbered CICIM Y0219, deposited at the Jiangnan University Industrial Microbial Resources and Information Center, CICIM Y0219) was used instead of the Loren Hidden Ball. The yeast Cryptococcus laurentii) (No. CICIM Y0232) was cultured in the same manner as in Example 5-1.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucose ceramide production promoter sample 5-1 of the present invention, and stored in a refrigerator at 4 ° C for use in cell culture. Additives.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged

(3000 rpm) 10 minutes, the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight as a sample of the ceramide/glucosylceramide production promoter of the present invention. 5-J, Sample 5-J was dissolved in 50% ethanol to prepare a 1% strength solution, and stored in a refrigerator at 4 °C as an additive for cell culture.

The ceramide/glucose ceramide production promoting effect of Samples 5-1 and 5-J was evaluated in the same manner as in Example 5-1, and the results are shown in Table 5. As shown in Table 5, it was confirmed that the sample 5-J of Example 5-4 had a glucose ceramide production promoting effect.

另外， 对于样品 5-A~5-J， 不将其加入到上述含角质化细胞的 12 孔板中 （即， 不进行上述细胞培养） 而直接迸行神经酰胺 /葡萄糖神经 酰胺量的分析，除此以外的操作与上述实施例 5- 1同样进行。结果确认 了样品 5-A〜5-J本身中没有神经酰胺或葡萄糖神经酰胺存在。

In addition, for the sample 5-A-5-J, it was not added to the above-mentioned 12-well plate containing keratinocytes (that is, the cell culture was not performed), and the amount of ceramide/glucosylceramide was directly analyzed. The other operations were carried out in the same manner as in the above Example 5-1. As a result, it was confirmed that there was no ceramide or glucosylceramide present in the sample 5-A to 5-J itself.

 This indicates that ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent. Example 6-1

(Manufacture of ceramide and/or glucosylceramide to produce a stimulating agent) As a microorganism for producing ceramide and/or glucosylceramide production promoter from bean dregs, Hansenula polymorpha) was purchased from Jiangnan University and deposited in CICIM, China Industrial Microbial Resources and Information Center, CICIM. Y0234 ) .

 The above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium at 30 Torr. The culture was carried out for 72 hours. The medium contained 10 w/v% okara and lw/v% glucose. Among them, the bean dregs are prepared by immersing the soybeans in water for 12 hours to sufficiently absorb the moisture of the soybeans, and then grinding and damaging the water-absorbing sun beans, filtering and removing the liquid components, and then draining the remaining residue to obtain.

 Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added, and the mixture was pulverized for 10 minutes by an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.), centrifuged at 3000 rpm for 30 minutes, and the supernatant was blown back. The supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide and/or glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 0.1 w/v% (the present invention 6-A-1), and then stored at 4 ° C for use in the subsequent Ceramide/glucose ceramide production promotion test. Example 6-2

As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Amenula polymorpha) (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0205) o The soybean residue fermentation extract was obtained in the same manner as in Example 6-1, and was stored as a ceramide and/or glucosylceramide production promoter (Inventive product 6-B-1) of the present invention, and stored at 4 ° C. For subsequent ceramide/glucose ceramide production promotion tests. Example 6-3 1 001877 As a microorganism for the production of ceramide/glucose ceramide production promoter from bean dregs, using Hansenula polymorpha iHansenula polymorph^ (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0257). The soybean residue fermentation extract was obtained in the same manner as in Example 6-1, and the ceramide and/or glucosylceramide production promoter of the present invention was further dissolved in 5 (%% ethanol) to adjust the concentration thereof. After 0.1 w/v% (inventive product 6-C-1), it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test. Example 6-4

 As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Haemda polymorpha is used (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0122) ). The soybean residue fermentation extract was obtained in the same manner as in Example 6-1 as a ceramide and/or glucosylceramide production promoter (Inventive product 6-D-1) of the present invention, and further, 50 ν / ν% ethanol was used. Dissolving the soybean residue fermentation extract and adjusting the concentration to lw/v%

(Inventive product 6-D-2), 0.1 w/v% (Inventive product 6-D-3), and stored at 4 ° C, was used for the subsequent ceramide/glucose ceramide production promoting test. Example 6-5

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Hansenula polymorpha) (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0135) . The soybean residue fermentation extract was obtained in the same manner as in Example 6-1, and was stored as a ceramide and/or glucosylceramide production promoter (Inventive product 6-E-1) of the present invention, and stored at 4 ° C, For subsequent ceramide/glucose ceramide production promotion experiments. Example 6-6

As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Hansenula sylvestris iHansenula ciferrii (purchased from Jiangnan University and deposited at the China University Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0150) was used. The soybean residue fermentation extract was obtained in the same manner as in Example 6-1 as a ceramide and/or glucosylceramide production promoter of the present invention, and further, the soybean residue fermentation extract was dissolved in 50 v/v% ethanol to adjust the concentration thereof. After lw/v% (product 6-F-1 of the present invention), it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test. Example 6-7

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, use Hansenula polymorpha Hans (la polymorpha) (purchased from Jiangnan University, and deposited in CICIM, China University Industrial Microbial Resources and Information Center, deposit number CICIM Y0197) o The soybean residue fermentation extract was obtained in the same manner as in Example 6-1, and was used as the ceramide and/or glucosylceramide production promoter (present invention 6-G-1) of the present invention, and further, 50 Wv% was used. Ethanol dissolves the bean dregs fermentation extract and adjusts the concentration to lw/v%

(Inventive product 6-G-2), 0.1 w/v% (inventive product 6-G-3), and stored at 4 ° C, was used for the subsequent ceramide/glucose ceramide production promoting test.

(ceramide/glucose ceramide production promotion test)

 <Keratinocytes culture >

First, activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium), and cells were cultured at 37 ° C and a CO ₂ concentration of 5%. The culture is maintained until the cell aggregation density (confluence) is 80 to 90%. Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at 1 × 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

Then, the medium containing no growth factor was replaced, and the aqueous solution of the soybean residue fermentation extract obtained in the above Examples 6-1 to 6-7 was added in an amount of 20 μl/well in the above-mentioned 12-well plate (n=2). . Separately, only 50 ν / ν% ethanol (without addition of ceramide and/or glucosylceramide production promoter) was added as a control, and culture was carried out for 72 hours under the same culture conditions. After 72 hours of culture, the supernatant culture solution was discarded, and the cells were washed twice with 2 ml of PBS (phosphate buffer) (2 ml/well), and then 1 ml of PBS was added to the wells, and the cells were collected by a cell harvester (cell shovel). Into the test tube for subsequent ceramide/glucose ceramide and protein analysis. <Lipid extraction>

 Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, methanol was added to the recovered cell-containing PBS solution: chloroform = 2.5 ml: 1.25 ml of a solvent, shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant and precipitation. Supernatant A was transferred to another tube for analysis of ceramide/glucose ceramide amount, and precipitate B was used for analysis of protein content.

<Protein Content Analysis>

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. Add 900 μl of SDS (sodium dodecyl sulfate) solution to the precipitate B recovered above, mix it, and heat-degrade the protein for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, add 2N HCl solution of ΙΟΟμΙ to it. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Assay Kit) method.

<Ceramid/Glucosylceramide Amount Analysis>

 To the remaining supernatant A, PBS: chloroform = 1.25 mh 1.25 ml of solvent was added, shaken for 20 minutes, centrifuged at 3000 rpm for 5 minutes, and the chloroform layer as the lower phase was separated and recovered, and protected under nitrogen at 30 ° C. The chloroform layer was dried for 40 minutes to obtain a dried product, and chloroform: methanol = 2.5 ml: 1.25 ml of a solvent was added, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and GlyCer, respectively). , the unit is g/ml) for quantification.

 Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9: v/v/v) as a layering agent for thin-layer chromatography, and the layering agent is applied to the top of the sheet to blow a layer of the layering agent with a hair dryer; Steps once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate on a baking plate at 180 ° C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucosyl ceramide).

Then, according to the following formula (1), the obtained values were respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The Ser/Pro and GlyCer/Pro of the reference are 1, The relative values of Cer/Pro and GlyCer/Pro of the present invention were determined. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Wdl and GlyCer/Well, relative amounts relative to the control). Also shown in Table 6.

 Formula 1 )

 Cer/Pro=Ceramic Amount (g/ml) / Protein Content (g/ml)

 GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content (g/ml) Table 6

如表 6所示，实施例 6-2中得到的本发明品既具有神经酰胺产生促 进效果， 又具有葡萄糖神经酰胺产生促进效果； 另外， 实施例 6-1、 实 施例 6-3以及实施例 6-6中得到的本发明品的神经酰胺产生促进效果显 著，而实施例 6-4以及实施例 6-7中得到的本发明品的葡萄糖神经酰胺 产生促进效果显著。 因此， 确认了本发明的神经酰胺和 /或葡萄糖神经 酰胺产生促进剂具有神经酰胺产生促进效果和 /或葡萄糖神经酰胺产生 促进效果。 (本发明的神经酰胺和 /或葡萄糖神经酰胺产生促进剂中不存在神 经酰胺和葡萄糖神经酰胺的试验）

As shown in Table 6, the inventive product obtained in Example 6-2 had both a ceramide production promoting effect and a glucosylceramide production promoting effect; Further, Example 6-1, Example 6-3, and Example The ceramide production-promoting effect of the present invention obtained in 6-6 was remarkable, and the glucosylceramide production-promoting effect of the present invention obtained in Example 6-4 and Example 6-7 was remarkable. Therefore, it was confirmed that the ceramide and/or glucosylceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect. (Test for the absence of ceramide and glucosylceramide in the ceramide and/or glucosylceramide production promoter of the present invention)

 The ceramide and/or glucosylceramide production promoter obtained in the above Examples 6-1 to 6-7 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide and glucosylceramide in the present invention. presence.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. . Example 7-1

 (Manufacture of ceramide and/or glucosylceramide production promoter)

 As a microorganism that produces a ceramide and/or glucosylceramide-producing agent from bean dregs, Bombyx serrata (Bulle tsugcw (purchased from Jiangnan University and preserved at CICIM, China University Industrial Microbial Resources and Information Center, preserved) No. CICIM Y0259 ) o

 The above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours. The medium contained 10 w/v% okara and l w/v% glucose. Among them, the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.

 Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. After diluting with 2.5 times of absolute ethanol, 1 ml was taken as a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 7-A).

Then, the crude product was treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifuged at 3000 rpm for 30 minutes, and the supernatant was recovered. Filter about 70 ml of the supernatant with a filter paper, and then dry the filtrate to a constant weight in a vacuum dryer below 40 ° C to obtain beans. The slag fermentation extract is used as a ceramide and/or glucosylceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 50 Wv% ethanol, and the concentration thereof was adjusted to 1 w/v% (inventive product 7-B), 0.1 w/v% (inventive product 7-C), and stored at 4°. C, for subsequent ceramide/glucose ceramide production promotion test.

(ceramide/glucose ceramide production promotion test)

 <Keratinocytes culture>

First, activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium), and cells were cultured at 37 ° C and a CO ₂ concentration of 5%. The culture is maintained until the cell aggregation density (concentration) is 80 to 90%. Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at 1 × 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced, and the aqueous solution of the soybean residue fermentation extract obtained in the above Example 7-1 was added in an amount of 20 μl/well in the above-mentioned 12-well plate (n=2). 7-A~7-C). In addition, only add 50v/V. /. Ethanol (without addition of ceramide and/or glucosylceramide production enhancer) was used as a control and cultured for 72 hours under the same culture conditions. After 72 hours of culture, the supernatant culture solution was discarded, and the cells were washed twice with 2 ml of PBS (phosphate buffer) (2 ml/well), and then 1 ml of PBS was added to the wells, and the cells were collected by a cell harvester (cell shovel). Into the test tube for subsequent ceramide/glucose ceramide and protein analysis.

<Lipid extraction>

 Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, methanol was added to the recovered cell-containing PBS solution: chloroform = 2.5 ml: 1.25 ml of a solvent, shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant and precipitation. Supernatant A was transferred to another tube for analysis of ceramide/glucose ceramide amount, and precipitate B was used for analysis of protein content.

<Protein Content Analysis> To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. Add 900 μl of SDS (sodium dodecyl sulfate) solution to the precipitate B recovered above, mix it, and heat-degrade the protein for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, add 2N HCl solution of ΙΟΟμΙ to it. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Assay Kit) method.

<Ceramid/Glucosylceramide Amount Analysis>

 To the remaining supernatant A, PBS: chloroform = L25 ml: 1.25 ml of a solvent, shaken for 20 minutes, centrifuged at 3000 rpm for 5 minutes, and the chloroform layer as a lower phase was separated and recovered, and protected under nitrogen at 30 ° C. The chloroform layer was dried for 40 minutes to obtain a dry product, and chloroform was added to ΙΟΟμΙ: methanol = 2.5 ml: 1.25 ml of a solvent, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and GlyCer, in g/ml), was quantified.

 Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9:1 v/v/v) as a layering agent for thin-layer chromatography, the layering agent is applied to the top of the sheet, and the layering agent is blown dry with a hair dryer; The above steps are once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate on a baking plate at 180 ° C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucosyl ceramide).

 Then, according to the following formula (1), the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The relative values of Cer/Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to be 100 (respectively referred to as Cer/Well and GlyCer/Well, respectively, relative to the relative amount of the control). Also shown in Table 7.

 Formula 1 )

 Cer/Pro=Ceramic Amount (g/ml) / Protein Content (g/ml)

 GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content (g/ml) Table 7

No. 对照品 1 1 1 1 实 本发明品 7-A 1.01 1.12 0.78 0.86 施 本发明品 7-B 1.74 2.03 1.07 1.26 例

No. Reference 1 1 1 1 Actual invention 7-A 1.01 1.12 0.78 0.86 Application of the invention 7-B 1.74 2.03 1.07 1.26 Example

 The present invention 7-C 4.23 3.95 0.96 0.90 7-1 As shown in Table 7, the ceramide production-promoting effect of the present invention 7-A to 7-C obtained in Example 7-1 was remarkable. Therefore, it has been confirmed that the ceramide and/or glucose amide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.

(Test for the absence of neuroamide and glucosylceramide in the ceramide and/or glucosylceramide production promoter of the present invention)

 The amide and/or glucosylceramide production promoter obtained in the above Example 7-1 was directly analyzed by TLC without cell culture, and the results showed that ceramide and glucosylceramide were not present in the present invention.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. . Example 8-1

 <Preparation of ceramide/glucosylceramide production promoter (fermentation liquid treatment) > First, the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. It is crushed, filtered and the liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentrations of the okara and glucose in the mixture were respectively 10% and 1 w/v%, and the mixture was used as a medium.

 Then, 100 ml of the above medium was added to an Erlenmeyer flask and inoculated with a certain amount of Rhodotonda mucilaginosa (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0250), and cultured at 30 °C. day.

The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and 1 ml of the mixture was taken as the ceramide of the present invention. Sample 8-A of the glucosylceramide production promoter was stored in a 4 C refrigerator for use as an additive for cell culture.

 The diluted ethanol solution was sonicated for 10 minutes, centrifuged at 80 rpm for 30 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight. The dried product was used as the sample of the ceramide and/or glucosylceramide production promoter of the present invention 8-B, 8-C, and the samples 8-B and 8-C were each dissolved in 50% ethanol to prepare 1%. A 0.1% strength solution was stored in a refrigerator at 4 ° C as an additive for cell culture. <Evaluation of ceramide/glucosylceramide production promoter>

 (cell culture)

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at 37 ° C and a CO ₂ concentration of 5%. The cells were cultured to a concentration of 80 to 90%. After passage, the cells were passaged into a 175 cm ² square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per cell at a cell concentration of 1×10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced in the above 12-well plate, and samples 8-A, 8-B, 8-C and a control (concentration of 5 (% by weight aqueous ethanol solution) were respectively added to the above 12-well plate. Medium (n=2), cultured for 3 days under the same culture conditions.

 After 3 days of culture, discard the supernatant medium, wash the cells twice with 2 ml of PBS (phosphate buffer), add 1 ml of PBS to the wells, and collect the cells into the tubes with a cell harvester (cell shovel). In order to prepare for subsequent ceramide/glucose ceramide and protein analysis.

 (Confirmation of ceramide/glucose ceramide production promoting effect)

 To the recovered cell-containing PBS solution, methanol: chloroform = 2.5 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant a and a precipitate b. The supernatant a was used for the analysis of the amount of ceramide/glucose amide, and the precipitate b was used for the analysis of the protein content.

(1) Analysis of ceramide/glucose ceramide amount To the supernatant a thus obtained, PBS: chloroform = 1.25 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain two upper and lower phases. The lower phase was recovered and dried under nitrogen at 40 ° C for 40 minutes. To the obtained dried product, chloroform of ΙΟΟ μ Ι was added: methanol = 2 mh 1 ml of a solvent was dissolved, and the amount of ceramide and glucosylceramide was measured by the following thin plate chromatography (TLC) method (respectively referred to as Cer and GlyCer, respectively). , the unit is g/ml).

 Place the ceramide (or glucosylceramide) standard and sample solution on a dry TLC plate, and perform thin-layer chromatography with CHCB : CH30H: CH3COOH ( 190: 9: 1 v/v/v) as a layering agent. The layer is applied to the top of the sheet and the spreader is blown dry with a blower; the above steps are repeated once. Then use CHCB: CH30H: C3H60 (76: 20: 4 v/v/v) as a layering agent for thin-layer chromatography, and wait for the layering agent to travel 2.5 cm from the bottom of the sheet, stop the layer, and blow dry with a hair dryer. . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatograph on a baking plate at 180 °C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucose ceramide).

 (2) Analysis of protein content

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. 90 μl of SDS (sodium dodecylsulfonate) solution was added to the above precipitate b and mixed, and the protein was denaturing and degraded by heating at 60 ° C for 2 hours, and after cooling, 2 μ of HCl solution of ΙΟΟμΙ was added thereto. The amount of protein (denoted as Pro, in g/ml) was determined according to the BCA Kit (Protein Quantitative Analysis Kit) method.

 (3) Analysis results

For the samples of the present invention and the control, Cer/Pro and GlyCer/Pro were respectively determined (see the following formula). The Ser/Pro and GlyCer Pro of the control were 1 and the relative values of Cer/Pro and GlyCer/Pro of the present invention are shown in Table 8. In addition, the relative value (Pro. (%)) of the amount of protein in each well of the present invention was determined by taking the amount of protein in each well of the reference product as 100, and the nerve of each well was determined accordingly. The amount of amide and glucosylceramide (relative to the relative amount of the control), ie, Cer/Well=Cer/Pro X Pro. (%), GlyCer/Well=GlyCer/Pro X Pro. (%) ₀ Table 8 shows.

If Cer/Pro (or Cer/Well) is greater than 1 (the value of the control), this sample has a ceramide production promoting effect; if GlyCer/Pro (or GlyCer/Well) is greater than 1 (the value of the reference), then This sample was shown to have a glucose ceramide production promoting effect. As shown in Table 8, it was confirmed that the inventive products 8-A, 8-B and 8-C have a ceramide production promoting effect. Example 8-2

 In the preparation of the ceramide/glucose ceramide production promoter of Example 8.1, the sticky red circle was replaced by Rhodotorula mucilaginosa ( deposited in the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0433). The yeast (Rhodotorula mucilaginosa ^) (No. CICIM Y0250) was cultured in the same manner as in Example 8.1.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 8-D of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to constant weight. Samples 8-E and 8-F which are ceramide/glucosylceramide production promoters of the present invention, and samples 8-E and 8-F were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture.

 The samples of the 8-D, 8-E, and 8-F ceramides were evaluated in the same manner as in Example 8-1.

/ Glucosylceramide production promoting effect, and the results are shown together in Table 8.

 As shown in Table 8, it was confirmed that the samples 8-E and 8-F of Example 8-2 had a glucose ceramide production promoting effect. Example 8-3

 In the preparation of the ceramide/glucose ceramide production promoter of Example 8.1, the viscosity was replaced by Rhodotorula mucilaginosa (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0430). The culture was carried out in the same manner as in Example 8-1 except that Rhodotonda mucilaginosa) (No. CICIM Y0250) was used.

The resulting culture was then centrifuged (3000 rpm, 10 minutes). Remaining supernatant The solution was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was used as the ceramide/glucose ceramide production promoter sample 8-G of the present invention, and stored in a refrigerator at 4 ° C for use as an additive for cell culture.

 The ceramide/glucose amide production promoting effect of the sample 8-G was evaluated in the same manner as in Example 8-1, and the results are shown in Table 8.

 As shown in Table 8, it was confirmed that the sample 8-G of Example 8-3 had a ceramide and glucose ceramide production promoting effect. Example 8-4

 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, R. erythromycin Rhodoto la glutinis (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0148) was used instead of the sticky red yeast. (Rhodotorula mucilaginosa) (No. CICIM Y0250) was cultured in the same manner as in Example 8.1.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and the diluted mixture of the above anhydrous ethanol was sonicated for 10 minutes, centrifuged (3000 rpm) for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered. The filtrate was filtered, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucosylceramide production promoter of the present invention. 8-H, and the sample 8-H was dissolved in 50% ethanol to prepare a concentration of 1%. The solution was stored in a refrigerator at 4 ° C as an additive for cell culture.

 The ceramide/glucose amide production promoting effect of the sample 8-H was evaluated in the same manner as in Example 8-1, and the results are shown together in Table 8.

 As shown in Table 8, it was confirmed that the sample 8-H of Example 8-4 had a glucosylceramide production promoting effect. Example 8-5

In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, the viscous red circle was replaced with the viscous red yeast iRhodotonda mucilaginosa} (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0324). Yeast Rhodotorula mucilaginos O (No. CICIM Y0250), in addition to Example 8-1 was also cultured in the same manner.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper. The filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer to obtain samples 8-1 and 8-J of the ceramide/glucosylceramide production promoter of the present invention, and samples 8-1 and 8-J were respectively 50%. The solution was dissolved in ethanol to prepare a solution having a concentration of 1% and 0.1%, and stored in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucosylceramide production promoting effects of the samples 8-1, 8-J were evaluated in the same manner as in Example 8-1, and the results are shown in Table 8.

 As shown in Table 8, it was confirmed that the samples 8-1 and 8-J of Example 8-5 had a glucosylamide-promoting effect. Example 8-6

 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, Rhodotoru mucilaginosa (preserved at Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0337) was used instead of viscous red. The culture was carried out in the same manner as in Example 8-1 except that Rhodotoruh mucilaginosa (No. CICIM Y0250) was used.

 The resulting culture was then centrifuged to GOOO rpm for 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 8-K of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

 The ceramide/glucose amide production promoting effect of the sample 8-K was evaluated in the same manner as in Example 8-1, and the results are shown together in Table 8.

 As shown in Table 8, it was confirmed that the sample 8-K of Example 8-6 had a glucose ceramide production promoting effect. Example 8-7

In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, Rhodotoruh mucilaginosa (reserved in Jiangnan University) The culture microbial resources and information center CICIM (No. CICIM Y0394) was cultured in the same manner as in Example 8-1 except that Rhodotorula muci ginosa (No. CICIM Y0250) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper. The filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucosylceramide production promoter of the present invention. 8-L, and the sample 8-L was dissolved in 50% ethanol to prepare a 1% concentration. The solution was stored in a refrigerator at 4 °C as an additive for cell culture.

 The ceramide/glucose amide production promoting effect of the sample 8-L was evaluated in the same manner as in Example 8-1, and the results are shown together in Table 8.

 As shown in Table 8, it was confirmed that the sample 8-L of Example 8-7 had a ceramide and glucose ceramide production promoting effect. Example 8-8

 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, R. sylvestris Rhodotoru mucilaginosa (preserved at Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIMY0418) was used instead of the viscous red yeast (Rhodotonda mucilaginosa (No. CICIM Y0250) was cultured in the same manner as in Example 8-1.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 8-M of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.

The mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to constant weight. Samples 8-N and 8-0 which are ceramide/glucosylceramide production promoters of the present invention, and samples 8-N and 8-0 were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture. The ceramide/glucose ceramide production promoting effect of the samples 8-M, 8-N, and 8-0 was evaluated in the same manner as in Example 8.1, and the results are shown in Table 8.

 As shown in Table 8, it was confirmed that the samples 8-M, 8-N, and 8-0 of Example 8-8 have a ceramide and glucosylceramide production-promoting effect. Example 8-9

 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, Rhodotorula mucilaginosa (preserved at Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0423) was used instead of viscous red. The culture was carried out in the same manner as in Example 8-1 except that Rhodotorula mucilaginoscO (No. CICIM Y0250) was used.

 The resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper. The filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer. Sample 8-P as a ceramide/glucosylceramide production promoter of the present invention, and sample 8-P was dissolved in 50% ethanol to prepare a 0.1% concentration. The solution was stored in a refrigerator at 4 ° C as an additive for cell culture.

 The ceramide/glucose amide production promoting effect of the sample 8-P was evaluated in the same manner as in Example 8-1, and the results are shown together in Table 8.

 As shown in Table 8, it was confirmed that the sample 8-P of Example 8-9 had a ceramide and glucose ceramide production promoting effect.

 Table 8

( Rhodotorula 本发明品 0.04 0.04 0.94 1.02 mucilaginosa ) 8-E

( Rhodotorula The present invention 0.04 0.04 0.94 1.02 mucilaginosa ) 8-E

 The invention is 0.05 0.07 0.85 1.1 1 8-F

 Sticky red round yeast

- CICIM The present invention

 ( Rhodotorula 1.20 1.13 1.24 1.16 Y0430 8-G

 Mucilaginosa )

 Red yeast

- CICIM The present invention

 ( Rhodotorula 0.44 0.43 1.06 1 .05 Y0148 8-H

 Glutinis )

 Invention product

 Sticky Red Round Yeast 0.24 0.28 1 .08 1.26- CICIM 8-1

 ( Rhodotorula

 Y0324 The present invention

 Mucilaginosa ) 0.57 0.47 1.50 1.23

 8-J

 Sticky red round yeast

- CICIM The present invention

 { Rhodotorula 0.45 0.41 1.69 1.54 Y0337 8-K

 Mucilaginosa)

 Sticky red round yeast

- CICIM The present invention

 ( Rhodotorula 3.19 2.48 4.62 3.60 Y0394 8-L

 Mucilaginosa )

 The invention product 4.27 3.20 3.51 2.63 8-M

 Sticky red round yeast

- CICIM The present invention

 ( Rhodotorula 2.06 1.77 2.59 2.23 Y0418 8-N

 Mucilaginosa )

 The invention is 2.41 1.94 2.52 2.03 8-0

 Sticky red round yeast

- CICIM The present invention

 ( Rhodotorula 1.96 2.12 2.38 2.58 Y0423 8-P

 Mucilaginosa ) In addition, for sample 8-A-8-P, the ceramide/glucosyl ceramide amount was directly analyzed without adding it to the above-mentioned 12-well plate containing keratinocytes (ie, without performing the above cell culture). The other operations were carried out in the same manner as in the above Example 8-1. As a result, it was confirmed that no ceramide or glucosylceramide was present in the samples 8-A to 8-P itself.

 This indicates that ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent. Example 9-1

(Manufacture of ceramide and/or glucosylceramide production promoter) As a microorganism that produces ceramide and/or glucosylceramide-producing agents from bean dregs, use Sporobolomyces msew) (purchased from Jiangnan University and preserved in CICIM, China University Industrial Microbial Resources and Information Center, under the accession number CICIM Y0247

 The above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours. The medium contained 10 w/v% okara and lw/v% glucose. Among them, the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.

 Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added to obtain a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 9-A), which was stored at 4 ° C, and was used for the subsequent ceramide/glucose nerve. Amide production promotion test. Example 9-2

 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Sporozolomium roseus ( purchased from Jiangnan University and preserved in CICIM of China University Industrial Microbial Resources and Information Center, with the accession number CICIM Y0193 Example 9-1 The crude product of the ceramide and/or glucosylceramide production promoter of the present invention was obtained in the same manner, and then the crude product was treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, and centrifuged at 3000 rpm for 30 minutes. The supernatant was recovered. The supernatant was filtered by a filter paper to a volume of about 70 ml, and the filtrate was dried in a vacuum dryer at 40 ° C or lower to a constant weight to obtain a bean slag as a ceramide/glucose ceramide production promoter of the present invention. Fermented extract.

Further, the soybean residue fermentation extract was dissolved in 5 (%% ethanol), adjusted to a concentration of 0.1 w/v% (inventive product 9-B), and stored at 4 ° C for subsequent ceramide/glucose nerves. Amide production promotion test. Example 9-3

 As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Sporozoimus spseus was purchased from Jiangnan University and preserved at the China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0132. The crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention product 9-C) was obtained in the same manner as in Example 9-1, and then treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.). The crude product was centrifuged for 10 minutes at 3000 rpm for 10 minutes, and the supernatant was recovered. The supernatant was filtered through a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% ethanol), adjusted to a concentration of 1 w/v% (inventive product 9-D), and stored at 4 ° C for the subsequent ceramide/glucosylceramide. Produce a boost test.

(ceramide/glucose ceramide production induces test)

 <Keratinocytes culture >

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at a concentration of 5% C0 ₂ at 37 ° C. The cell culture was carried out under the conditions until the cell converence was 80 to 90%. Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at IX 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced, and the aqueous solution of the soybean residue fermentation extract obtained in the above Examples 9-1 to 9-3 was added to the above-mentioned 12-well plate (n=2) in an amount of 20 μl/well. . In addition, only 5 (^% ethanol (without adding ceramide/glucose ceramide production promoter) was added as a control, and cultured for 72 hours under the same culture conditions. After 72 hours of cultivation, the supernatant culture solution was discarded.

The cells were washed twice with 2 ml PBS (phosphate buffer), 1 ml PBS was added to the wells, and cells were collected into a tube with a cell harvester (cell shovel) for subsequent ceramide/glucose ceramide and protein analysis. <Lipid extraction>

 Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, methanol was added to the recovered cell-containing PBS solution: chloroform = 2.5 ml: 1.25 ml of a solvent, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant. And precipitation. Supernatant A was transferred to an additional tube for analysis of the amount of ceramide/glucose amide, and precipitate B was used for analysis of protein content.

<protein content analysis>

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. Add 900 μl of SDS (sodium dodecyl sulfate) solution to the precipitate B recovered above, mix it, and heat-degrade the protein for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, add 2N HCl solution of ΙΟΟμΙ to it. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Assay Kit) method.

<Ceraamide/Glucosylceramide Amount Analysis>

 To the remaining supernatant A, PBS: chloroform = 1.25 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, centrifuged at 3000 rpm for 5 minutes, and the chloroform layer as a lower phase was separated and recovered, and nitrogen gas was at 30 ° C. The dried chloroform layer was protected for 40 minutes to obtain a dried product, which was dissolved in chloroform: methanol = 2.5 ml: 1.25 ml of a solvent, and the amount of ceramide and glucosylceramide was determined by the following thin plate chromatography (TLC) method (respectively Quantify for Cer and GlyCer in g/ml).

 Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9:1 v/v/v) as a layering agent for thin-layer chromatography, and the layering agent is applied to the top of the sheet to blow a layer of the layering agent with a hair dryer; The above steps are once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate on a 180-inch baking plate for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucosyl ceramide).

Then, according to the following formula (1), the obtained values were respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The Ser/Pro and GlyCer/Pro of the reference are 1, The relative values of Cer/Pro and GlyCer/Pro of the present invention were determined. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Wdl and GlyCer/Well, relative amounts relative to the control). Also shown in Table 9.

 Formula 1 )

 Cer/Pro = amount of amide (g/ml) / protein content (g/ml)

 GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content (g/ml) Table 9

如表 9所示， 实施例 9-1、 实施例 9-3中得到的本发明品的葡萄糖 神经酰胺产生促进效果显著，而实施例 9-2中得到的本发明品的神经酰 胺产生促迸效果显著。

As shown in Table 9, the glucosylceramide production-promoting effect of the present invention obtained in Example 9-1 and Example 9-3 was remarkable, and the ceramide production of the present invention obtained in Example 9-2 was promoted. The effect is remarkable.

 Therefore, it was confirmed that the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect. .

(Test for the absence of ceramide and glucosylceramide in the ceramide/glucosylceramide production promoter of the present invention)

 The ceramide/glucosylceramide production promoter obtained in the above Examples 9-1 to 9-3 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide or glucosylceramide present in the present invention.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. . Example 10-1

(Manufacture of ceramide and/or glucosylceramide production promoter) As a microorganism that produces a ceramide and/or glucosylceramide-producing agent from bean dregs, use Bretonnomyces clausseni (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, preservation number) CICIM Y0125

 The above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours. The medium contained 10 w/v% okara and lw/v% glucose. Among them, the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.

 Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added, followed by treatment with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifugation at 3000 rpm for 30 minutes, and the supernatant was recovered. The supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried in a vacuum dryer at 40 ° C or lower to constant weight to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 5 (% ethanol), adjusted to a concentration of 1 w/v% (inventive product 10-A), and stored at 4 ° C for the subsequent ceramide/glucose nerve. Amide production promotion test. Example 10-2

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, use the Brassica yeast iBrettanomyces clausseniO (purchased from Jiangnan University and preserved in CICIM, China University Industrial Microbial Resources and Information Center, under the accession number CICIM Y025 1 An okara fermentation extract which is a ceramide/glucose ceramide production promoter of the present invention was obtained in the same manner as in Example 10-1.

Further, the soybean residue fermentation extract was dissolved in 50% by weight of ethanol, adjusted to a concentration of 0.1 w/v% (inventive product 10-B), and stored at 4 ° C for subsequent ceramide/glucosylceramide Produce a boost test. Example 10-3

 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, use Bremennomyces claussenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0127) o The soybean residue fermentation extract which is a ceramide/glucose ceramide production promoter of the present invention was obtained in the same manner as in Example 10-1.

 Further, the soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 10_C) and 0.1 w/v% (inventive product 10-D), and then stored in 4 At °C, it was used in subsequent ceramide/glucose ceramide production promotion tests.

(ceramide/glucose ceramide production promotion test)

 <Keratinocytes culture >

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at 37 °C.

The cell culture was carried out under the condition that the concentration of C0 ₂ was 5% until the cell aggregation density (confluence) was 80 to 90%. Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at IX 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

 Then, the medium containing no growth factor was replaced, and the aqueous solution of the soybean residue fermentation extract obtained in the above Examples 10-1 to 10-3 was added to the above-mentioned 12-well plate (n=2) in an amount of 20 μl/well. . Further, only 50 ν / ν% ethanol (without adding a ceramide / glucosylceramide production promoter) was added as a control, and culture was carried out for 72 hours under the same culture conditions. After 72 hours of culture, discard the supernatant culture solution.

 The cells were washed twice with 2 ml PBS (phosphate buffer), and 1 ml of PBS was added to the wells, and the cells were collected into a test tube by a cell harvester (cell shovel) for subsequent ceramide/glucose ceramide and protein analysis.

<Lipid extraction>

Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, a solvent of methanol: chloroform = 2.5 m 1.25 ml is added to the recovered cell-containing PBS solution. The mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant and a precipitate. Supernatant A was transferred to an additional tube for analysis of ceramide/glucose ceramide amount and Precipitate B was used for analysis of protein content. <protein content analysis>

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. Add 900 μl of SDS (sodium dodecyl sulfate) solution to the precipitate B recovered above, mix it, and heat-degrade the protein for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, add 2N HCl solution of ΙΟΟμΙ to it. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Assay Kit) method.

<Ceramid/Glucosylceramide Amount Analysis>

 To the remaining supernatant A, PBS: chloroform = 1.25 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, centrifuged at 3000 rpm for 5 minutes, and the chloroform layer as a lower phase was separated and recovered, and nitrogen gas was at 30 ° C. The dried chloroform layer was protected for 40 minutes to obtain a dried product, which was dissolved in chloroform: methanol = 2.5 mh in 1.25 ml of solvent, and the amount of ceramide and glucosylceramide was determined by the following thin plate chromatography (TLC) method (respectively as Cer and GlyCer, in g/ml) were quantified.

 Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9:1 v/v/v) as a layering agent for thin-layer chromatography, the layering agent is applied to the top of the sheet, and the layering agent is blown by a blower; Repeat the above steps once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate on a baking plate at 180 ° C for 7 minutes to develop color. The chromogenic TLC plate was scanned and analyzed for ceramide (or glucosyl ceramide).

Then, according to the following formula (1), the obtained values were respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The relative values of Cer/Pro and GlyCer Pro of the present invention were determined by using Ser/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 10. Formula 1)

 Cer/Pro=Ceramic Amount (g/ml) / Protein Content (g/ml)

 GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content g/ml) Table 10

如表 10所示，实施例 10-1中得到的本发明品既具有神经酰胺产生 促进效果， 又具有葡萄糖神经酰胺产生促进效果， 而实施例 10-2、 实 施例 10-3中得到的本发明品的葡萄糖神经酰胺产生促进效果显著。

As shown in Table 10, the present invention obtained in Example 10-1 has both a ceramide production promoting effect and a glucosylceramide production promoting effect, and the examples obtained in Examples 10-2 and 10-3. The glucosylceramide production promoting effect of the invention product is remarkable.

 Therefore, it was confirmed that the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.

(Test for the absence of ceramide and glucosylceramide in the ceramide/glucosylceramide production promoter of the present invention)

 The ceramide/glucose ceramide production promoter obtained in the above Examples 10-1 to 10-3 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide and glucosylceramide present in the present invention.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. . Example 11-1

 (Manufacture of ceramide and/or glucosylceramide production promoter)

As a microorganism for producing ceramide and/or glucosylceramide production promoter from bean dregs, rr _OW a lipolytica ( purchased from Jiangnan University and deposited in CICIM, China University Industrial Microbial Resources and Information Center, is used. Deposit number CICIM Y0311)o The above-mentioned strain obtained by culturing in YPD (Yeast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium at 30 Torr. The culture was carried out for 72 hours. The medium contains 10 w/v% okara and ^% glucose. Among them, the bean dregs are prepared by soaking the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.

 Then, in order to recover the ceramide and/or glucosylceramide production promoter from the obtained culture solution, the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added, followed by treatment with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifugation at 3000 rpm for 30 minutes, and the supernatant was recovered. The supernatant was filtered through a filter paper to a thickness of about 70 m, and the filtrate was dried in a vacuum dryer at 40 ° C or lower to a constant weight to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.

 Further, the soybean residue fermentation extract was dissolved in 50 v/v% ethanol, adjusted to a concentration of 1 w/v%, and stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.

(ceramide/glucose ceramide production promotion test)

 <Keratinocytes culture >

First, normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm ² flask (2 ml of growth factor-containing medium) at a concentration of 5% C0 ₂ at 37 ° C. The cell culture was carried out under the conditions until the cell aggregation density (concentration) was 80 to 90 ° /. . Cells were passaged to 175cm ² flasks side cultured cells to a concentration of 80~90%. Then, it was passaged to a 12-well plate (2 ml per well) at 1 × 10 ⁵ cells/ml, and the cells were further cultured to a concentration of 80 to 90%.

Then, the medium containing no growth factor was replaced, and the aqueous ethanol solution of the soybean residue fermentation extract obtained in the above Example 11-1 was added in an amount of 20 μl/well in the above-mentioned 12-well plate (n=2). Further, only 50 ν / ν% ethanol (without addition of a ceramide/glucose ceramide production promoter) was added as a control, and culture was carried out for 72 hours under the same culture conditions. After 72 hours of culture, discard the supernatant culture solution. The cells were washed twice with 2 ml PBS (phosphate buffer), 1 ml PBS was added to the wells, and cells were collected into a tube with a cell harvester (cell shovel) for subsequent ceramide/glucose ceramide and protein analysis. <Lipid extraction>

 Lipid extraction was performed using the Bligh & Dyer method. Specifically, first, a solvent containing methanol: chloroform = 2.5 m 1.25 ml was added to the recovered cell-containing PBS solution, and the mixture was shaken for 20 minutes, and centrifuged for GOOO rpm for 5 minutes to obtain a supernatant and a precipitate. . Supernatant A was transferred to an additional tube for analysis of the amount of ceramide/glucose amide, and precipitate B was used for analysis of protein content.

<protein content analysis>

 To characterize the amount of ceramide/glucosylceramide in the cells, protein content analysis was performed. Add 900 μl of SDS (sodium dodecyl sulfate) solution to the precipitate B recovered above, mix it, and heat-degrade the protein for 2 hours in a water bath at 60 ° C for 2 hours. After cooling, add 2N HCl solution of ΙΟΟμΙ to it. The amount of protein (denoted as Pro, in g/ml) was determined by the BCA Kit (BCA Protein Concentration Assay Kit) method.

<Ceramid/Glucosylceramide Amount Analysis>

 To the remaining supernatant A, PBS: chloroform = 1.25 m 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, centrifuged at 3000 rpm for 5 minutes, and the chloroform layer as a lower phase was separated and recovered, and protected at 30 ° C with nitrogen gas. The dried chloroform layer was dried for 40 minutes to obtain a dried product, which was dissolved in chloroform: methanol = 2.5 ml: 1.25 ml of a solvent, and the amount of ceramide and glucosylceramide was determined by the following thin plate chromatography (TLC) method (respectively Cer and GlyCer, in g/ml) were quantified.

Using a dry TLC plate, using a chloroform:methanol:acetic acid (190:9:1 v/v/v) as a layering agent for thin-layer chromatography, the layering agent is applied to the top of the sheet, and the layering agent is blown dry with a hair dryer; The above steps are once. Then use chloroform: methanol: acetone (76: 20: 4 v / v / v) as a layering agent for thin-layer chromatography, to the layering agent to 2.5cm from the bottom of the thin plate, stop the layer, blow dry with a hair dryer . Spray with copper sulfate phosphoric acid solution, blow dry, and place the chromatography plate at 180 ° C Bake on the baking sheet for 7 minutes to develop color. The chromogenic TLC plate was scanned and subjected to ceramide (or glucosylceramide) analysis.

 Then, according to the following formula (1), the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro. The relative values of Cer/Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 11.

 Formula 1 )

 Cer/Pro-ceramide amount (g/ml) / protein content (g/ml)

如表 11所示，实施例 11-1中得到的本发明品的葡萄糖神经酰胺产 生促进效果显著。 GlyCer/Pro=Glucose ceramide amount (g/ml) / protein content ( μ _δ /πι1 ) Table 1 1

As shown in Table 11, the glucosylceramide production-promoting effect of the present invention obtained in Example 11-1 was remarkable.

 Therefore, it was confirmed that the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.

(Test for the absence of ceramide and glucosylceramide in the ceramide/glucosylceramide production promoter of the present invention)

 The amide/glucosylceramide production promoter obtained in the above Example 11-1 was directly analyzed by the TLC method without cell culture, and the results showed that there was no ceramide and glucosylceramide present in the present invention.

 This indicates that the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. .

Claims

Rights request A method for producing a ceramide and/or glucosylceramide production promoter, wherein  The yeast is cultured in a medium containing okara and sugar, and a ceramide production promoter and/or a glucosylceramide production promoter is obtained from the culture supernatant. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 1, wherein  The yeast is selected from the group consisting of the yeast Schizosaccharomyces, Pichia (Pichia ) is a yeast, Candida (Ccmdkia yeast, Debaryomyces yeast, Cryptococcus iCryptococcus yeast, Hansenula (H listen enula) yeast, B. spp. iBullercO yeast And at least any one of the genus Rhodotorula (Rhodotorula yeast, S. sporobolomyces yeast, Brettanomyces yeast, and Yarrowia (rrm «) yeast. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The Schizosaccharomyces genus Schizosaccharomyces yeast (Schizosaccharomyces octoporus ). The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The Pichia genus Pichia yeast is Pichia membranifaciens or Pichia membranifaciens or P. sylvestris Pichia norgus (Pichia norvegensis) or Pichia membranifaciens or Pichia burtonii. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein The Candida yeast Candida boidinii, Candida parapsilosis > Candida magnoliae > Maltose Candida maltose Candida steatolytica, Candida butyri, Candida rhagi O or Candida metapsilosis. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The Debaryomyce yeast is Desmondella hansenii ( Debaryomyces hansenii ) or Fan Wei] β德巴禾 [J yeast ( Debaryomyces vanrijiae ) ₀ The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein The genus Cryptococcus (Cryptococc) the yeast is Cryptococcus laurentii (Cryptococcus laurentii) ¹ J Wo Hungary or Cryptococcus (Cryptococcus hungaricus). The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The Hansenula yeast is Ha emda polymorpha or Hansenula ciferrif. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The B. cerevisiae Bidle yeast is the spore yeast of the genus Hemlock, E. tsugae. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein The Rhodotorula yeast is Rhodotonda mucilaginosa or Rhodotorula glutinis The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The sporozoite ( Spowbohmyce yeast is Sporozolomidae roseus The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The Brettcmomyce yeast is Brettanomyces claussenii. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 2, wherein  The Yarrowia (rrow) yeast is rraW lipolytica. The method for producing a ceramide and/or glucose amide amide production promoter according to any one of claims 1 to 13, wherein  The saccharide is glucose. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 14, wherein  The medium consists of bean dregs, glucose and water. The method for producing a ceramide and/or glucosylceramide production promoter according to claim 15, wherein  The content of the bean dregs in the medium is 8 to 12 w/v%, and the content of the saccharides is 0.8 to 1.2 w/v%. 17. The ceramide and/or glucose god according to any one of claims 1 to 16. a method for producing an amide production promoter, wherein  The cultivation was carried out at 27 to 35 °C. The method for producing a ceramide and/or glucose amide amide production promoter according to any one of claims 1 to 17, wherein  The culture time is 1 to 7 days. The method for producing a ceramide and/or glucose amide amide production promoter according to any one of claims 1 to 18, wherein  The culture obtained by the culture was centrifuged to obtain the supernatant. The method of producing a ceramide and/or glucosylceramide production promoter according to claim 19, wherein  After the ethanol is added to the supernatant obtained after centrifugation, it is used as a ceramide production promoter and/or a glucosylceramide production promoter. A method of producing a ceramide and/or glucosylceramide production promoter according to claim 20,  Further, the substance obtained by adding the ethanol is further purified by ultrasonic treatment, centrifugation, filtration, and vacuum drying to obtain a ceramide and/or a glucosylceramide production promoter. 22. The use of a fermented extract of okara for promoting the production of ceramide and/or glucosylceramide,  The fermented extract of the bean dregs is obtained by culturing yeast in a medium containing okara. 23. Use of a fermented extract of bean dregs as a ceramide production promoter and/or a glucosamine amide production promoter, The fermented extract of the bean dregs is obtained by culturing yeast in a medium containing okara. 24. Use of a fermented extract of okara to produce a ceramide production promoter and/or a glucose amide production promoter,  The fermented extract of the bean dregs is obtained by culturing yeast in a medium containing okara. The use according to any one of claims 22 to 24, wherein  The yeast is selected from the group consisting of Schizosaccharomyces yeast, Pichia yeast, Candida (Candida yeast, DeBalibacterium iDebaryomyces) yeast, Cryptococcus yeast, Hansenula Ha enula yeast, B. spp. (Bull yeast, Rhodotorula yeast, Saccharomyces sp.) (Sporobo myces yeast, Brassica genus yeast, Arroyos (rro '«) yeast At least any one of the yeasts. 26. The use according to claim 25, wherein  The Schizosaccharomyces genus iSchizosaccharomyces yeast is Schizosaccharomyces octoporus. 27. The use according to claim 25, wherein  The Pichia yeast is Pichia anomala, Pichia guilliermondii, Pichia norvegensis, Pichia membranifaciens or Burton. Pick ia b urton ii. 28. The use according to claim 25, wherein The Candida yeast Candida zeylanoides, Candida boidinii, Candida parapsilosis Candida magnoliae > Maltose silk Yeast iCandidamaltoscd, Candida candida steatolytic Candida butyri~, Candida rhagi or Candida metapsilosis. 29. The use according to claim 25, wherein  The Debaryomyce yeast is Debaryomyces hansenii or Debaryomyces vanrijiae. 30. The use according to claim 25, wherein The genus Cryptococcus (Cryptococcus) the yeast is Cryptococcus laurentii (Cryptococcus laurentii) ¹ J Wo Hungary or Cryptococcus (Cryptococcus hungaricus). The use according to claim 25, wherein  The Hansenula (H ms ula ) yeast is Hansenula polymorpha or Hansenula ciferrii. 32. The use according to claim 25, wherein  The Bullerella genus Bullera yeast is the spores of the genus Hemoptera Bi kra tsugae ) o 33. The use according to claim 25, wherein  The Rhodoton a yeast (Rhodotonula mucilaginosa or Rhodotorula glutinis). 34. The use according to claim 25, wherein  The sporophyte ί Sporobohmyce yeast is a pink throwing yeast ( Sporobolomyces roseus ). 35. The use according to claim 25, wherein  The Brassica genus Bretta yces yeast is Brettanomyces claussenii. 36. The use according to claim 25, wherein The Rraw yeast is rraw lipolytica