JP2013534136A - Method for producing ceramide and / or glucosylceramide production promoter - Google Patents

Abstract

The present invention relates to a method for producing a ceramide and / or glucosylceramide production promoter, wherein yeast is cultured in a medium containing soybean residue and saccharide, and ceramide and / or glucosylceramide production promoter is obtained from the culture supernatant. I will provide a.
SOLUTION: A ceramide and / or glucosylceramide production promoter is efficiently obtained at low cost by using a specific microorganism and using an easily available soybean residue as a main raw material of a medium.
[Selection figure] None

Description

  The present invention relates to a method for producing a ceramide and / or glucosylceramide production promoter capable of promoting the production of ceramide and / or glucosylceramide in the skin. In particular, the present invention uses ceramide and / or ceramide and / or ceramide and / or glucosylceramide production that can promote the production of ceramide / glucosylceramide in the skin by using a specific microorganism and using soybean residue, which is a by-product of the food processing industry, as a main raw material of the medium. The present invention relates to a method for producing a glucosylceramide production promoter.

  The skin has the ability to produce ceramide and glucosylceramide. In the skin, particularly in the horny stratum corneum, ceramide synthesized and secreted by epidermal cells accumulates and is the main component of intercellular lipids. Ceramide, one of the sphingolipids, is present in the stratum corneum of the skin and occupies about 50% of intercellular lipids in the stratum corneum. Sphingolipid is a general term for complex lipids containing a sphingosine skeleton. Glucosylceramide is a kind of sphingolipid in which glucose is bound to ceramide. Ceramide synthesized in skin epidermal cells is temporarily stored in the form of glucosylceramide or sphingomyelin, then discharged to the outside of the cell, and the action of β-glucocerebrosidase and sphingomyelinase In response, it becomes ceramide again and functions as an intercellular lipid. Ceramide and glucosylceramide in the skin have physiological effects such as improving the skin moisturizing function and the skin barrier function.

  Conventionally, it is known that sphingolipids such as ceramide, glucosylceramide, galactosylceramide, etc. enhance the water retention ability of the stratum corneum and are effective in improving rough skin (Patent Documents 1 and 2). In addition, the skin has the ability to produce ceramide / glucosylceramide, but recently, the skin tends to become dry due to the influence of environment, climate, and the like. Therefore, the ability to improve the skin moisturizing function by producing the skin's own ceramide and the like is not sufficient.

  Therefore, a method of replenishing the skin with ceramide from the outside can be considered. For example, Patent Document 3 describes a method for producing glucosylceramide by culturing Candida tropicalis JPCCY0004 strain (NITE P-570) belonging to the genus Candida. The moisture retaining function of the skin can be improved by applying the glucosylceramide to human dry skin. However, since the method is not related to the ceramide / glucosylceramide production promoter, it is merely a method for directly obtaining glucosylceramide present in a large amount of cells after culturing, so ceramide of glucosylceramide synthesized by that method The skeleton is different from the ceramide skeleton of ceramides existing in the human body because the double bond exists at two positions and has a branch. Therefore, when such a glucosylceramide obtained directly from the body of a microorganism is applied to human skin, it cannot be applied to the skin, and there is a risk of causing problems related to safety in use.

  In addition, the method of replenishing the skin with ceramide from the outside also has a problem that it is insufficient in terms of the durability of the moisturizing effect, as in the case of conventionally used moisturizing agents. Further, depending on the state of the skin, it may be insufficient to absorb ceramide or the like supplemented from the outside, and the moisturizing effect may not be sufficiently exhibited.

  On the other hand, if the skin's own ability to produce ceramide or glucosylceramide can be enhanced or promoted, there is no need to supplement ceramide or glucosylceramide from the outside, and the skin itself produces ceramide and / or glucosylceramide, thereby barriering the skin. It is considered that the function and moisture retention function can be improved. Furthermore, since the ceramide and / or glucosylceramide obtained in this way is produced from the skin itself, the ceramide having the structure of normal skin is synthesized and the safety such as poor adaptability to the skin. It is thought that the sex problem will disappear.

  Therefore, recently, research on producing substances aimed at promoting the production of epidermal ceramide in the stratum corneum of the skin has been actively promoted. Here, as a substance that can promote the generation of ceramide, ceramide production promoters containing an extract such as mannentake, bee pollen, and senkyu as active ingredients have been reported (Patent Documents 4 to 6). However, all of these ceramide production promoters are extracted from plant raw materials, and this extraction operation takes a long time, and the availability of natural resources is limited, so that the production cost is high.

  Patent Document 7 discloses a ceramide synthesis promoter containing a fungus culture containing nicotinic acid and / or nicotinamide, which can activate ceramide synthesis of epidermal cells themselves inside the skin surface layer, It is disclosed that the fungal culture may be a lactic acid bacteria culture, a bifidobacteria culture, a mushroom cell culture, or a yeast culture. However, from the examples of Patent Document 7, the amount of ceramide produced in lactic acid bacteria cultures or yeast cultures (Comparative Examples 1 to 4) that do not contain nicotinic acid and / or nicotinamide is significantly different from the control. I understand that there is no. Therefore, the ceramide synthesis promoter contains nicotinic acid and / or nicotinamide as a substantial active ingredient, and since the ceramide / glucosylceramide production promoting effect cannot be said to be sufficient, there is room for improvement. The medium used for preparing the ceramide synthesis promoter substantially contains nicotinic acid and / or nicotinamide as an active ingredient, so it can be said that the production cost is high.

  Yeast is a unicellular fungus widely distributed in nature, grows in an acidic environment mainly containing moist sugar, and can survive in an oxygen-deficient environment. Yeast can be used for the production of brewing. Conventionally, more than 1000 types of yeast have been known, including Schizosaccharomyces yeast, Pichia genus yeast, Candida genus yeast, Debaryomyces genus yeast, Cryptococcus genus yeast, Hansenula genus yeast, Bullera genus yeast, Rhodotorula genus Yeast of the genus Sporobolomyces, yeast of the genus Brettanomyces, and yeast of the genus Yarrowia.

  The genus Schizosaccharomyces produces alcohol by fermentation and has been used for food processing for a long time, and has also been used as a biological research material.

  Moreover, the yeast of the genus Pichia has the following properties and uses.

  Yeast cells of the genus Pichia are spherical, elliptical, and long, sometimes have a cone shape, and do not just form vertices. Asexual breeding methods can grow for multi-sided buds, and some types can form nodules. In general, an individual ascomy contains 1-4 ascospores, and occasionally contains more than 4 ascospores. The spores are hat-shaped, hemispherical or spherical with horns. After maturation, the sac usually rips and a small number do not rip, forming a sac without joining or joining. Junctions occur between female cells and buds or between two independent cells. Both mycelia and pseudomycelia are ascombs, but their form does not become large or form a spindle, and they are mixed in one or different gates. Pichia yeasts can assimilate nitrates and are negative in the DBB reaction.

  The position regarding the classification of Pichia is Ascomycota-Hemiascomycetes-Saccharomycetales-Saccharomycetaceae-Pichia.

  As a use of yeast of the genus Pichia, for example, methanol can be used to produce useful substances such as antibiotics and biological proteins; or it can be used as a genetic engineering fungus. Pichia pastoris is used for the production of reduced glutathione and other parts of the yeast of the genus Pichia are used for the production of specific proteins / enzymes.

  In addition, microorganisms belonging to the genus Candida are known to have the following uses. For example, Candida valida is used for lipase production. Candida metapsilosis, Candida utilis, etc. can also be used in the production of brews, glycerin, edible yeast, organic acids and enzyme agents, and the feed industry. In addition, microorganisms belonging to the genus Candida are useful for the treatment of waste and the production of edible proteins in the industrial and agricultural by-product processing industries.

  In addition, it is known that microorganisms belonging to Debaryomyces are capable of producing D-arabitol by fermenting glucose or converting xylose into xylitol in general industrial applications.

  Studies on microorganisms of the genus Cryptococcus are also being conducted. For example, it has been reported that mutant cryptococcus produces β-galactosidase. Furthermore, Cryptococcus laurentii has an inhibitory effect on the storage of Geotrichum citri-aurantii.

  Also, since most of the microorganisms belonging to the genus Hansenula can produce ethyl acetate and enhance the scent of the product, it is usually known that it is used in sake brewing and the food industry. .

  In addition, it is known that microorganisms belonging to the genus Bullera are generally used in fermented foods such as fermented sausages.

  Studies on microorganisms of the genus Rhodotorula are also being conducted. For example, some species of the genus Rhodotorula have the effect that they can be weakly oxidized to hydrocarbons and can synthesize β-carotene. For example, Rhodotorula glutinis can oxidize alkanes to produce 50-60% fat in dry biomass. Under certain conditions, α-alanine and glutamic acid can be produced, methionine production capacity is strong, and the production amount can be 1% of dry biomass.

  In addition, microorganisms belonging to the genus Sporobolomyces are known to be industrially useful yeasts such as accumulating coenzyme Q10 in the cells. In addition, microorganisms belonging to the genus Sporobolomyces are also attracting attention as producers of useful substances such as carotenoids and L-carnitine.

  Microorganisms belonging to the genus Brettanomyces are a kind of yeast and are widely present in nature. It is well known to grow during barrel storage to produce ethylphenol. Generally, it is hated because it has a bad influence on the aroma of beer and wine.

  It is well known that microorganisms belonging to the genus Yarrowia such as Yarrowia lipolytica are particularly useful for the production of polyunsaturated fatty acids “PUFA”.

  However, it has not been reported yet that a production promoter for ceramide or glucosylceramide is produced by a yeast (particularly, yeast of each genus described above).

JP-A 61-260008 JP-A 61-271205 JP 2010-22217 A JP 2005-194240 A JP 2010-70499 A JP 2010-150237 A JP-A-9-194383

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

  As a result of intensive studies to solve the above problems, the present inventor has found that ceramide and / or glucosylceramide production promoter can be efficiently obtained at low cost by culturing yeast in a medium containing soybean residue and saccharide. The present invention has been made.

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

  The present invention also relates to the use of a fermented extract of soybean residue obtained by culturing yeast in a medium containing soybean residue and saccharides for promoting production of ceramide and / or glucosylceramide.

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

  The present invention also relates to the use of a fermented extract of soybean residue obtained by culturing yeast in a medium containing soybean residue and saccharides for the production of a ceramide and / or glucosylceramide production promoter.

  In the present invention, the yeast is Schizosaccharomyces yeast, Pichia yeast, Candida yeast, Debaryomyces yeast, Cryptococcus yeast, Hansenula yeast, Bullera yeast, Rhodotorula yeast, Sporobolomyces yeast. It may be at least one selected from yeast, Brettanomyces genus yeast, and Yarrowia genus yeast.

  According to the production method of the present invention, a ceramide and / or glucosylceramide production promoter can be inexpensively and efficiently used by using, as a main raw material of a medium, a specific microorganism that is easily available such as soybean residue. Can be obtained. Since the said promoter can improve the ceramide and / or glucosylceramide production ability which skin has efficiently, it can also improve the moisture retention function of skin effectively.

  The production method of the present invention is to cultivate yeast in a medium containing soybean residue and saccharide, and obtain a ceramide and / or glucosylceramide production promoter from the culture supernatant.

(Microorganisms that can be used for the production of ceramide and / or glucosylceramide production promoter)
Microorganisms that can be used for the production of the ceramide and / or glucosylceramide production promoter of the present invention include yeast of the genus Schizosaccharomyces, yeast of the genus Pichia, yeast of the genus Candida, yeast of the genus Debaryomyces, yeast of the genus Cryptococcus, yeast of the genus Hansenula It may be at least one selected from yeast, yeast of the genus Bullera, yeast of the genus Rhodotorula, yeast of the genus Sporobolomyces, yeast of the genus Brettanomyces, and yeast of the genus Yarrowia.

  Examples of microorganisms belonging to the genus Schizosaccharomyces that can be used in the present invention include Schizosaccharomyces octoporus and Schizosaccharomyces pombe, which are commonly used in the food industry. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included.

  Of these, Schizosaccharomyces octoporus is preferred from the viewpoint that the resulting product has a higher ceramide and / or glucosylceramide production promoting effect.

  Examples of microorganisms belonging to the genus Pichia that can be used in the present invention include Pichia anomala, Pichia guilliermondii, Pichia norvegensis, Pichia membranifaciens, Pichia burtonii, Pichia farinose, and Pichia Kluyveri that are commonly used in the food industry. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included.

  Among these, Pichia anomala, Pichia guilliermondii, Pichia norvegensis, Pichia membranifaciens, and Pichia burtonii are preferable from the viewpoint that the obtained product has a higher ceramide and / or glucosylceramide production promoting effect.

  Pichia membranifaciens is also referred to as Pichia membranaefaciens, Pichia alcoholophila, Pichia hyalospora, Pichia scaptomyzae, Willia belgica or Zygopichia guilliermondii.

  Pichia burtonii is also referred to as Candida fibrae, Dematium chodati, Endomycopsis chodati, Hyphopichia burtonii or Trichosporon behrendii.

  As a microorganism belonging to the genus Candida that can be used in the present invention, used in the food field, Candida zeylanoides, Candida boidinii, Candida parapsilosis, Candida magnoliae, Candida maltosa, Candida steatolytica, Candida butyri, Candida rhagii, Candida metapsirosis (Candida metapsilosis), Candida albicans, Candida holmii, or at least one microorganism selected from Candida valida. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among them, from the viewpoint that the agent obtained in the present invention has a ceramide production promoting effect and / or a glucosylceramide production promoting effect, preferably Candida zeylanoides, Candida boidinii, Candida parapsilosis, Candida magnoliae, Candida maltosa, Candida steatolytica (also known as Candida hellenical), Candida butyri ), Candida rhagii or Candida metapsilosis.

  The microorganism belonging to the genus Debaryomyces that can be used in the present invention is selected from Debaryomyces hansenii, Debaryomyces polymorphus or Debaryomyces vanrijiae used in the food field. At least one kind of microorganism. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among them, since the agent obtained in the present invention has a ceramide production promoting effect and / or a glucosylceramide production promoting effect, it is preferably Debaryomyces hansenii or Debaryomyces vanrijiae.

  Examples of microorganisms belonging to the genus Cryptococcus that can be used in the present invention include Cryptococcus laurentii, Cryptococcus hungaricus, and Cryptococcus terreus, which are commonly used in the food industry. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included.

  Among them, Cryptococcus laurentii and Cryptococcus hungaricus are preferable from the viewpoint that the obtained product has a higher ceramide and / or glucosylceramide production promoting effect.

  Examples of microorganisms belonging to the genus Hansenula that can be used in the present invention include Hansenula polymorpha, Hansenula ciferrii, Hansenula subpelliculosa, Hansenula subpelliculosa, Examples thereof include at least one microorganism selected from satanus (Hansenula saturnus), Hansenula anomala (Hansenula anomala), and the like. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among them, since the agent obtained in the present invention has a ceramide production promoting effect and / or a glucosylceramide production promoting effect, it is preferably Hansenula polymorpha (also known as Pichia angusta, Hansenula). • Annsta (Hansenula angusta), Ogataea polymorpha (Ogataea polymorpha), or Hansenula ciferrii.

  Microorganisms belonging to the genus Bullera that can be used in the present invention are selected from the group used in the food field, such as Bullera tsugae, Bullera variabilis, and Bullera albus. There may be at least one microorganism. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among them, since the agent obtained in the present invention has a ceramide production promoting effect and / or a glucosylceramide production promoting effect, it is preferably Bullera tsugae (also known as Sporobolomyces tsugae). ).

  Examples of microorganisms belonging to the genus Rhodotorula that can be used in the present invention include Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula minuta, Rhodotorula pallida, and Rhodotorula rubra. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included.

  Of these, Rhodotorula mucilaginosa and Rhodotorula glutinis are preferred from the viewpoint that the resulting product has a higher ceramide and / or glucosylceramide production promoting effect.

  Examples of microorganisms belonging to the genus Sporobolomyces that can be used in the present invention include Sporobolomyces roseus, Sporobolomyces salmonicolor, and Sporobolomyces luber (Sporobolomyces rosemon) used in the food field. Sporobolomyces ruber) and the like. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among these, since the agent obtained by the present invention has a ceramide production promoting effect and / or a glucosylceramide production promoting effect, Sporobolomyces roseus is preferable.

  Examples of the microorganism belonging to the genus Brettanomyces used in the present invention include at least one microorganism used in the field of food and selected from Brettanomyces claussenii or Brettanomyces anomalus. It is done. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among them, since the agent obtained in the present invention has a ceramide production promoting effect and / or a glucosylceramide production promoting effect, Brettanomyces claussenii is preferable.

  Examples of microorganisms belonging to the genus Yarrowia that can be used in the present invention include mutants obtained from microorganisms such as Yarrowia lipolytica or Yarrowia lipolytica based on conventional methods. The microorganism exhibits an action of culturing in a medium containing soybean residue and saccharide, thereby obtaining an agent that improves the ability of epidermal cells to produce ceramide and / or glucosylceramide from the culture supernatant. Among them, since the agent obtained in the present invention has a ceramide production promoting effect and / or glucosylceramide production promoting effect, preferably Yarrowia lipolytica (also known as Candida lipolytica), Candida paralipolytica, Saccharomycopsis lipolytica, Endomycopsis lipolytica, and Pseudomonilia deformans.

  All of the microorganisms (yeasts) according to the present invention are purchased through commercial routes such as the China Common Microorganisms Storage Center (CGMCC) and the China Traditional Culture Storage Center (CCTCC), the Gangnam University Industrial Microbial Resources and Information Center (CICIM), etc. It can be done.

  Moreover, in the manufacturing method of this invention, the said microorganisms (yeast) can be used individually by 1 type or in combination of 2 or more types.

(Method for producing ceramide and / or glucosylceramide production promoter)
In the method for producing a ceramide and / or glucosylceramide production promoter of the present invention, ceramide and / or glucosylceramide production promoter is obtained from the culture supernatant by culturing yeast in a medium containing soybean residue and saccharide. Details of the method are described below.

<Medium>
In the present invention, it is desirable to culture at least one or more of the above microorganisms in a liquid medium.

  The medium used in the present invention contains soybean residue and saccharides as main components. Since soy residue and saccharides are readily available raw materials, the medium of the present invention has an advantage that is superior in terms of cost compared to the prior art. In addition, for example, 1% peptone, 0.5% yeast powder and the like may be added as appropriate.

  Among them, the soybean residue is obtained by immersing soybean in water, sufficiently absorbing water, pulverizing and filtering, removing water, and drying. It is also possible to use the soy residue left in the process of producing soy products that have been squeezed soymilk in the food industry.

  Examples of the saccharide include sucrose, maltose, and sucrose. Any one of them can be used alone or in combination. Sucrose can be suitably used from the viewpoint of enhancing fermentation productivity and availability and improving the effect of promoting ceramide and / or glucosylceramide production.

  In the present invention, from the viewpoint of enhancing the effect of promoting ceramide and / or glucosylceramide production, the medium is preferably composed of soybean residue, sucrose and water. Among them, soybean residue is 5 to 20% (w / v), more preferably 8 to 12% (w / v), and sucrose is 0.2 to 2% (w / v), more preferably 0.8 to It is preferable to contain 1.2% (w / v).

<Culture conditions>
This is carried out by directly inoculating the microbial cells in the ceramide / glucosylceramide production promoter production medium. Culture conditions for producing a ceramide and / or glucosylceramide production promoter include physiological characteristics of yeast microorganisms, optimization of temperature and time required for the growth of yeast microorganisms, and promotion of ceramide and / or glucosylceramide production. From the viewpoint of enhancing the effect, it is preferable to culture at a culture temperature of 20 to 40 ° C., preferably 27 to 35 ° C., for 1 to 7 days, preferably 48 to 96 hours, more preferably 68 to 76 hours.

<Recovery and purification of ceramide and / or glucosylceramide production promoter>
From the viewpoint of increasing the purity of the product, the culture solution obtained by the culture is purified to obtain a supernatant. As a purification method, commonly used operations such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, and crystallization may be appropriately combined as necessary. Moreover, it is preferable to obtain a supernatant by centrifugation. The number of rotations of centrifugation is preferably 2000 to 4000 rpm, more preferably 2500 to 3500 rpm. Centrifugation time is preferably 0 to 20 minutes, and more preferably 5 to 15 minutes.

  Specifically, ethanol may be added to the supernatant obtained by centrifugation to sterilize it, and the ceramide and / or glucosylceramide production promoter of the present invention may be used.

  In addition, the bacterial cells are removed from the culture solution by centrifugation, followed by sonication, and centrifugation to recover the supernatant. Again, the collected supernatant is filtered to remove impurities and the like, and further dried in a vacuum to obtain the ceramide and / or glucosylceramide production promoter of the present invention.

  The fermented extract of soybean residue obtained by culturing the yeast of the present invention and obtained from the culture supernatant has an action of increasing the amount of ceramide and / or glucosylceramide in normal human keratinocytes.

  Therefore, the fermented extract of soybean residue obtained by culturing the yeast of the present invention and obtained from the culture supernatant can be used for therapeutic or non-therapeutic purposes for promoting ceramide and / or glucosylceramide production. It can also be used as a glucosylceramide production promoter for therapeutic or non-therapeutic purposes. Non-therapeutic uses include cosmetic use and use for maintaining health. In addition, the above-mentioned therapeutic purpose use and non-therapeutic purpose use can be performed separately.

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

  The ceramide and / or glucosylceramide production promoter of the present invention increases the ceramide and / or glucosylceramide in the stratum corneum, and restores and improves the barrier function and moisturizing function of the skin, quasi-drugs, cosmetics Etc. can also be used. In addition, the ceramide or glucosylceramide production promoter can be used as a quasi-drug or cosmetic product with the concept of promoting ceramide or glucosylceramide production promotion, as necessary.

  The use form of the ceramide and / or glucosylceramide production promoter of the present invention can be used as an additive for addition to cultured cells, and can be blended into skin external preparations such as skin cleansing agents and makeup cosmetics. You can also. For example, it can be provided in various forms such as lotion, emulsion, gel, cream, ointment, powder, granule and the like. The base of the external preparation may be a known external base and is not particularly limited. In order to prepare such various external preparations for skin, the ceramide and / or glucosylceramide production promoter of the present invention is used alone or in combination with external preparations for skin such as skin cleansing agents and makeup cosmetics. Oily ingredients, moisturizers, powders, pigments, emulsifiers, solubilizers, UV absorbers, thickeners, medicinal ingredients, fragrances, antibacterial and fungicides, plant extracts, alcohols, etc. Can do.

  When the ceramide and / or glucosylceramide production promoter of the present invention is added to the cultured epidermal cell system to promote ceramide and / or glucosylceramide synthesis, the addition amount is 0.0001 to 10 w as a dried product of the promoter. / v%, more preferably 0.001 to 5 w / v%. When the addition amount is 0.0001 w / v% or more, the production promotion effect is sufficient. When the addition amount is 10 w / v% or less, it is preferable in that the stimulation to epidermal cells is small.

  The blended amount of the ceramide and / or glucosylceramide production promoter of the present invention into the external preparation for skin is a dry product of the fermented extract of soybean residue, which sufficiently promotes the synthesis of ceramide and / or glucosylceramide to moisturize the skin. Is effective, and the color and odor of the culture is less likely to occur, so that it is preferably 0.01 to 20 w / v%, more preferably 0.1 to 10 w / v% of the whole skin external preparation. is there.

  EXAMPLES Next, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to these.

(Example 1-1)
<Manufacture of ceramide and / or glucosylceramide production promoter (treatment of fermentation broth)>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.

  Next, 100 ml of the above medium was placed in an Erlenmeyer flask, inoculated with Schizosaccharomyces octoporus (Gangnam University Industrial Microbial Resources and Information Center (consigned by CICIM as number CICIM Y0123)) in a prescribed amount and cultured at 30 ° C. for 3 days .

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is used as samples 1-A and 1-B of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 1-A and 1-B are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.

  Next, the medium containing no growth factor was changed in the 12-well plate, and the product of the present invention (Samples 1-A, 1-B) and the control (50 v / v% ethanol solution) were respectively added to the 12-well plate (n = 2). ). The cells were cultured for 3 days under the same conditions.

  Thereafter, the culture medium of the supernatant was removed, and the cells were washed twice with PBS (2 mL / Well). Then, 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

(Confirmation of ceramide / glucosylceramide promoting effect)
First, methanol: chloroform (2.5 ml: 1.25 ml) was added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) was performed, and the supernatant a and the precipitate b were separated. The supernatant a was subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate b was subjected to the next protein amount analysis.

(1) Analysis of the amount of ceramide and the amount of glucosylceramide PBS: chloroform (1.25 ml: 1.25 ml) is added to the supernatant a, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and in the lower phase The chloroform layer was isolated and collected. The recovered chloroform layer was dried at 30 ° C. in a nitrogen atmosphere. The dried product was dissolved in 100 μl of a mixed solvent of chloroform / methanol (volume ratio = 2.5 ml: 1.25 ml) and subjected to quantification of ceramide and glucosylceramide by the following thin layer chromatography.

  A ceramide (or glucosylceramide) standard product and a sample solution were placed in a dry TLC plate, and thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. . After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide) (Cer and GlyCer, respectively, unit μg / mL).

(2) Analysis of protein amount In order to display the amount of ceramide and the amount of glucosylceramide in cells, the amount of protein was analyzed. To the precipitate b, a 900 μl SDS (sodium dodecyl sulfonate) solution was added, mixed, heated at 60 ° C. for 2 hours, and then the protein was denatured and decomposed and cooled. Subsequently, 100 μl 2N HCl was added, and the protein amount (denoted as Pro. Μg / mL) was measured by the BCA Kit method (protein amount analysis kit).

(3) Results of Analysis Cer / Pro and GlyCer / Pro were measured for each of the product of the present invention and the control sample as shown in the following formula. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated and shown in Table 1 when each of Cer / Pro and GlyCer / Pro of the control was 1. In addition, the relative value (Pro. (%)) Of the amount of protein per well of the present invention when the amount of protein per well of the control is 100 was calculated, and thereby the amount of ceramide and glucosylceramide per well were calculated. The amount (relative to the control) was determined. The results are also shown in Table 1.
Cer / Well = Cer / Pro × Pro. (%)
GlyCer / Well = GlyCer / Pro × Pro. (%)

  In addition, when Cer / Pro (or Cer / Well) is higher than 1 (control), it is considered that the sample has a ceramide production promoting effect. When GlyCer / Pro (or GlyCer / Well) is higher than 1 (control), the sample is considered to have a glucosylceramide production promoting effect.

  As is apparent from Table 1, the products 1-A and 1-B of the present invention were confirmed to have an effect of promoting production of ceramide and / or glucosylceramide.

  For samples 1-A to 1-B, the amount of ceramide and glucosylceramide were not added to the 12-well plate containing human activated keratinocytes (ie, the cell culture was not performed). Quantity analysis was performed. Except for the above, the procedure was the same as Example 1-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any of samples 1-A to 1-B.

  From this, ceramide or glucosylceramide does not exist in the product itself of the present invention, but the product of the present invention has an effect of promoting ceramide and / or glucosylceramide production, and a ceramide and / or glucosylceramide production promoter. Can be used as

(Example 2-1)
<Manufacture of ceramide and / or glucosylceramide production promoter (treatment of fermentation broth)>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.

  Next, 100 ml of the above medium was placed in an Erlenmeyer flask, and Pichia anomala (Gangnam University Industrial Microbial Resources and Information Center (consigned to CICIM as number 360-20-3)) was inoculated in a prescribed amount, and 3 at 30 ° C. Cultured for days.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 volumes of EtOH are added, and 1 ml of the mixed solution is used as the ceramide and / or glucosylceramide production promoter (sample 2-A) of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes were seeded in 2 ml each in a 12-well plate (1 × 10 ⁵ cells / mL) and continuously cultured until reaching 80-90% confluence.

  Next, the medium containing no growth factor was changed in the 12-well plate, and the product of the present invention (Sample 2-A) and control (50 v / v% ethanol solution) were added to the 12-well plate (n = 2). . The cells were cultured for 3 days under the same conditions.

  Thereafter, the culture medium of the supernatant was removed, and the cells were washed twice with PBS (2 mL / Well). Then, 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

(Confirmation of ceramide / glucosylceramide promoting effect)
To the PBS solution containing the collected cells, first, a solvent of methanol: chloroform (2.5 ml: 1.25 ml) was added and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) was performed, and the supernatant a and the precipitate b were separated. The supernatant a was subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate b was subjected to the next protein amount analysis.

(1) Analysis of the amount of ceramide and the amount of glucosylceramide PBS: chloroform (1.25 ml: 1.25 ml) solvent is added to the supernatant a, vibrated for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the lower phase The chloroform layer in was isolated and recovered. The recovered chloroform layer was dried at 30 ° C. in a nitrogen atmosphere. The dried product was dissolved in 100 μl of a mixed solvent of chloroform / methanol (volume ratio = 2.5 ml: 1.25 ml) and subjected to quantification of ceramide and glucosylceramide by the following thin layer chromatography.

  A ceramide (or glucosylceramide) standard product and a sample solution were placed in a dry TLC plate, and thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. . After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide) (Cer and GlyCer, respectively, unit μg / mL).

(2) Analysis of protein amount In order to display the amount of ceramide and the amount of glucosylceramide in cells, the amount of protein was analyzed. To the precipitate b, a 900 μl SDS (sodium dodecyl sulfonate) solution was added, mixed, heated at 60 ° C. for 2 hours, and then the protein was denatured / decomposed and cooled. Subsequently, 100 μl 2N HCl was added, and the protein amount (denoted as Pro. Μg / mL) was measured by the BCA Kit method (protein amount analysis kit).

(3) Results of Analysis Cer / Pro and GlyCer / Pro were measured for each of the product of the present invention and the control sample as shown in the following formula. Tables 2-1 to 2-3 show the relative values of Cer / Pro and GlyCer / Pro of the present invention when each of Cer / Pro and GlyCer / Pro of the control is set to 1. In addition, the relative value (Pro. (%)) Of the protein amount per well of the present invention when the amount of protein per well of the control is 100 was calculated, whereby the amount of ceramide and glucosyl per well were calculated. The amount of ceramide (relative to the control) was determined. The results are also shown in Table 2.
Cer / Well = Cer / Pro × Pro. (%)
GlyCer / Well = GlyCer / Pro × Pro. (%)

  In addition, when Cer / Pro (or Cer / Well) is higher than 1 (control), it is considered that the sample has a ceramide production promoting effect. When GlyCer / Pro (or GlyCer / Well) is higher than 1 (control), the sample is considered to have a glucosylceramide production promoting effect.

  As apparent from Tables 2-1 to 2-3, the product 2-A of the present invention was found to have an effect of promoting production of ceramide and glucosylceramide.

(Example 2-2)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala was designated as Pichia guilliermondii (Gangnam University Industrial Microbial Resources and Information Center (CICIM under the number 422-19-3). In addition, the mixture obtained by adding 2.5 volumes of EtOH to the supernatant obtained after fermentation of the soybean residue was treated with an sonicator for 10 minutes, and centrifuged at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a certain weight. Glucosylceramide production promoters 2-B and 2-C were used, and dried samples 2-B and 2-C were dissolved in 50 v / v% EtOH to give concentrations of 1 v / v% and 0.1 v / v%, respectively. After preparing a solution of The sample was stored in a refrigerator and subjected to the next cell culture, except for the above, in the same manner as in Example 2-1, the effect of the ceramide and / or glucosylceramide production promoter of Sample 2-B, 2-C was evaluated. The results are shown in Tables 2-1 to 2-3.
As is clear from Tables 2-1 to 2-3, the products 2-B and 2-C of the present invention were confirmed to have a ceramide production promoting effect.

(Example 2-3)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala is Pichia norvegensis (Gangnam University Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM Y0151). The culture was carried out in the same manner as in Example 2-1, except that

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as sample 2-D of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-E and 2-F of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-E and 2-F were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-D, 2-E, and 2-F was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-D, 2-E, and 2-F of Example 2-3 exhibited a ceramide production promoting effect.

(Example 2-4)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia anomala (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0297.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 volumes of EtOH are added, and 1 ml of the mixed solution is used as a sample 2-G of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as sample 2-H of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 2-H was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-G and 2-H was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-G and 2-H of Example 2-4 exhibited a ceramide and glucosylceramide production promoting effect.

(Example 2-5)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia anomala (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0349.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixture is used as a sample 2-I of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 2-J and 2-K of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 2-J and 2-K are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-I, 2-J, and 2-K was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Table 2-1 to Table 2-3, it was confirmed that Samples 2-I, 2-J, and 2-K of Example 2-5 exhibited a ceramide and / or glucosylceramide production promoting effect. It was.

(Example 2-6)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia membranifaciens (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was performed in the same manner as in Example 2-1, except that it was changed to Y0360.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 volumes of EtOH is added, and 1 ml of the mixed solution is made into 2-L of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 2-M and 2-N of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-M and 2-N are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-L, 2-M, 2-N was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-L, 2-M, and 2-N of Example 2-6 exhibited a ceramide production promoting effect.

(Example 2-7)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia anomala (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0420.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as sample 2-O of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 2-P and 2-Q of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 2-P and 2-Q are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-O, 2-P, and 2-Q was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Table 2-1 to Table 2-3, it was confirmed that Samples 2-O, 2-P, 2-Q of Example 2-7 exhibited a ceramide production promoting effect.

(Example 2-8)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia anomala (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0421.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixture is used as sample 2-R of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 2-S and 2-T of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-S and 2-T are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-R, 2-S, and 2-T was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-R, 2-S, and 2-T of Example 2-8 exhibited a ceramide production promoting effect.

(Example 2-9)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia burtonii (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0424.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 volumes of EtOH is added, and 1 ml of the mixed solution is used as a sample 2-U of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as sample 2-V of the ceramide and / or glucosylceramide production promoter of the present invention, and sample 2-V was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-U, 2-V was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-U and 2-V of Example 2-9 exhibited a ceramide production promoting effect.

(Example 2-10)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2, Pichia anomala (No. 360-20-3) was designated as Pichia guilliermondii (Gangnam University Industrial Microbial Resources and Information Center (CICIM as CICIM Y0440). The culture was carried out in the same manner as in Example 2-1, except that it was changed to the contract.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as a sample 2-W of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-W was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Table 2-1 to Table 2-3, it was confirmed that Sample 2-W of Example 2-10 exhibited a glucosylceramide production promoting effect.

(Example 2-11)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was assigned to Pichia guilliermondii (Gangnam University Industrial Microbial Resources and Information Center CICIM No. CICIM Y0326). The culture was carried out in the same manner as in Example 2-1, except that it was changed to.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixed solution is used as ceramide and / or glucosylceramide production promoter sample 2-X of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as sample 2-Y of the ceramide and / or glucosylceramide production promoter of the present invention, and sample 2-Y is further dissolved in 50 v / v% EtOH to obtain a solution having a concentration of 0.1 v / v%. After the preparation, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-X, 2-Y was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-X and 2-Y of Example 2-11 exhibited a glucosylceramide production promoting effect.

(Example 2-12)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia guilliermondii (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0329.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 volumes of EtOH are added, and 1 ml of the mixture is used as sample 2-Z of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as sample 2-a of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 2-a was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-Z, 2-a was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Tables 2-1 to 2-3, it was confirmed that Samples 2-Z and 2-a of Example 2-12 exhibited a ceramide and / or glucosylceramide production promoting effect.

(Example 2-13)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, Pichia anomala (No. 360-20-3) was changed to Pichia guilliermondii (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM The culture was carried out in the same manner as in Example 2-1, except that it was changed to Y0414.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as sample 2-b of the ceramide and / or glucosylceramide production promoter of the present invention, and sample 2-b was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-b was evaluated. The results are shown in Tables 2-1 to 2-3.

  As is clear from Table 2-1 to Table 2-3, it was confirmed that Sample 2-b of Example 2-13 exhibited a ceramide and glucosylceramide production promoting effect.

  Note that samples 2-A to 2-Z and 2-a, 2-b are not added to the 12-well plate containing the human activated keratinocytes (ie, the cell culture is not performed). The amount of ceramide and the amount of glucosylceramide were analyzed as they were. Except for the above, the procedure was the same as in Example 2-1. As a result, it was confirmed that neither ceramide nor glucosylceramide was present in any of samples 2-A to 2-Z and 2-a, 2-b.

  From this, ceramide or glucosylceramide does not exist in the product itself of the present invention, but the product of the present invention has an effect of promoting ceramide and / or glucosylceramide production, and a ceramide and / or glucosylceramide production promoter. Can be used as

(Example 3-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Candida zeylanoides (Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and the original number 472- 20-1) was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. To obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention (product 3-A-1 of the present invention).

  Further, the crude extract was treated with an ultrasonic treatment apparatus (manufactured by Branson) for 10 minutes, and the supernatant was recovered by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1 v / v% (the product 3-A-2 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 3-2)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Candida zeylanoides (Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and the original number 406- 20-1) was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-B-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-B-2) and 0.1 v / v% (present product 3-B-3). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-3)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Candida parapsilosis (Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and the original number 514- 20-3) was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-C-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1 v / v% (the product 3-C-2 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 3-4)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Candida parapsilosis (Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and the original number 525- 20-2) was used, and the fermented extract of soybean residue was used as the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-D-1) and 0.1 v / v% (present product 3-D-2). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-5)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Candida metapsilosis (Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and the original number 510- 19-3) was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-E-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-E-2) and 0.1 v / v% (present product 3-E-3). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-6)
Candida parapsilosis (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0314 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-F-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (the present invention product 3-F-2) and 0.1 v / v% (the present invention product 3-F-3). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-7)
Candida parapsilosis (purchased from Gangnam University as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and contract number CICIM Y0322 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Invention product 3-G-1). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-G-2) and 0.1 v / v% (present product 3-G-3). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-8)
Candida boidinii (Candida boidinii), a microorganism that produces ceramide and / or glucosylceramide production promoters from soybean residues, purchased from Gangnam University and commissioned by China High School Industrial Microbial Resources and Information Center CICIM Y0366 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-H-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1 v / v% (the product 3-H-2 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 3-9)
Candida metapsilosis (Purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0439 The fermented extract of soybean residue was obtained in the same manner as in Example 3-1.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-I-1) and 0.1 v / v% (present product 3-I-2) of After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-10)
Candida parapsilosis (Purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0443 The fermented extract of soybean residue was obtained in the same manner as in Example 3-1.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-J-1) and 0.1 v / v% (present product 3-J-2). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-11)
Candida magnoliae (manufactured from Cangida magnoliae) as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, and contract number CICIM Y0184) was used to obtain a fermented extract of soybean residue in the same manner as in Example 3-1.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-K-1) and 0.1 v / v% (present product 3-K-2). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-12)
Candida maltosa (manufactured as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, purchased from Candida maltosa (Gangnam University) and commissioned by China High School Industrial Microbial Resources and Information Center CICIM Y0207 The fermented extract of soybean residue was obtained in the same manner as in Example 3-1.

  Furthermore, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, adjusted to a concentration of 0.1 v / v% (the product 3-L-1 of the present invention), stored at 4 ° C., and then the next ceramide And / or subjected to a glucosylceramide production promotion test.

(Example 3-13)
Candida steatolytica (manufactured as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, purchased from Candida steatolytica (Gangnam University) and commissioned by China High School Industrial Microbial Resources and Information Center CICIM Y0221 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-M-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract is used as the ceramide and / or glucosylceramide production promoter of the present invention and stored at 4 ° C. to promote the production of the following ceramide and / or glucosylceramide. Take the test.

(Example 3-14)
Candida butyri (Candida butyri), a microorganism that produces ceramide and / or glucosylceramide production promoters from soybean residues, purchased from Gangnam University and commissioned by China High School Industrial Microbial Resources and Information Center, CICIM Y0233 The fermented extract of soybean residue was obtained in the same manner as in Example 3-1.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, adjusted to a concentration of 1 v / v% (the product 3-N-1 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 3-15)
Candida rhagii (Purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0248 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Product 3-O-1 of the present invention). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and 1 v / v% (present product 3-O-2) and 0.1 v / v% (present product 3-O-3). After preparation to a concentration, it is stored at 4 ° C., and is used for the next ceramide and / or glucosylceramide production promotion test.

(Example 3-16)
Candida zeylanoides (Candida zeylanoides), a microorganism that produces ceramide and / or glucosylceramide production promoters from soybean residue, purchased from Gangnam University and commissioned by China High School Industrial Microbial Resources and Information Center, CICIM Y0428 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Invention product 3-P-1). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, adjusted to a concentration of 1 v / v% (the product 3-P-2 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 3-17)
Candida parapsilosis (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0325 ) Was used to obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 3-1 (Invention product 3-Q-1). Furthermore, the fermented extract of soybean residue obtained by purifying the crude extract was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1 v / v% (product 3-Q-2 of the present invention), stored at 4 ° C., and then subjected to the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Culture of keratinocytes>
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Next, the medium containing no growth factor was changed in the 12-well plate, and 20 μl / well of an ethanol solution of the fermented soybean residue obtained in Examples 3-1 to 3-17 was added. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken and mixed for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated in a 60 ° C. water bath for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, mixed by shaking for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. . The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. These are also shown in Tables 3-1 to 3-2.

  As shown in Table 3-1 to Table 3-2, the products of the present invention obtained in Example 3-1, Example 3-6, 3-7 and Example 3-17 are ceramide production promoting effects and glucosylceramide production. The promotion effect was shown. In addition, the products of the present invention obtained in Example 3-2, Examples 3-9 to 3-13 and Example 3-15 showed a remarkable glucosylceramide production promoting effect. The products of the present invention obtained in Examples 3-3 to 3-5, Example 3-8, Example 3-14, and Example 3-16 showed a significant ceramide production promoting effect.

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have the effect of promoting production of ceramide and / or glucosylceramide.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Examples 3-1 to 3-17 above was analyzed only by the TLC method without performing the cell culture, and as a result, the products of the present invention include It was shown that ceramide / glucosylceramide was not present.

  From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

(Example 4-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
As a microorganism for producing a ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Debaryomyces hansenii (Gangnam University) and commissioned by China High School Industrial Microbial Resources and Information Center CICIM. -1) was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. Furthermore, it processed for 10 minutes with the ultrasonic processing apparatus (made by Branson), and supernatant was collect | recovered by 3000 rpm and 30 mins centrifugation. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-2)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Debaryomyces hansenii (Gangnam University) and commissioned by China High School Industrial Microbial Resources and Information Center CICIM. The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-3)
Debaryomyces hansenii (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, and commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0356) The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-4)
Debaryomyces hansenii (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0286) The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-5)
Debaryomyces hansenii (purchased from Gangnam University as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0287) The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-6)
Debaryomyces hansenii (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0289) The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-7)
Debaryomyces hansenii (purchased from Gangnam University as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0290) The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-8)
Debaryomyces vanrijiae (Debaryomyces vanrijiae), a microorganism that produces ceramide and / or glucosylceramide production promoters from soybean residues, purchased from Gangnam University and commissioned by China High School Industrial Microbial Resources and Information Center, CICIM Y0198 The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 0.1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Example 4-9)
Debaryomyces hansenii (Debaryomyces hansenii), a microorganism that produces ceramide and / or glucosylceramide production promoters from soybean residues, purchased from Gangnam University and commissioned by China High School Industrial Microbial Resources and Information Center, CICIM Y0351 The fermented extract of soybean residue obtained by the same method as in Example 4-1 was used as the ceramide and / or glucosylceramide production promoter of the present invention.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Keratinocytes culture>
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Next, the medium containing no growth factor in the 12-well plate was changed, and 20 μl / well each of ethanol solutions of 1% w / v content soybean fermented extract of the above Examples 4-1 to 4-9 was used. Added. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated at 60 ° C. for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. The results are also shown in Table 4.

  As shown in Table 4, the products of the present invention obtained in Example 4-1 and Example 4-9 showed a ceramide production promoting effect and a glucosylceramide production promoting effect. The products of the present invention obtained in Example 4-2 and Examples 4-4 to 4-7 showed a remarkable ceramide production promoting effect, but the present invention obtained in Example 4-3 and Example 4-8. The product showed a remarkable glucosylceramide production promoting effect.

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have a ceramide and / or glucosylceramide production promotion effect.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Examples 4-1 to 4-9 was analyzed only by the TLC method without performing the cell culture, and as a result, the products of the present invention include It was shown that ceramide / glucosylceramide was not present.
From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

(Example 5-1)
<Manufacture of ceramide and / or glucosylceramide production promoter (treatment of fermentation broth)>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.

  Next, 100 ml of the above medium was placed in an Erlenmeyer flask, inoculated with Cryptococcus laurentii (Gangnam University Industrial Microbial Resources and Information Center (consigned by CICIM as number CICIM Y0232)) in a prescribed amount, and cultured at 30 ° C. for 3 days. .

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as sample 5-A of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 5-B and 5-C of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 5-B and 5-C were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.

  Next, the medium containing no growth factor was changed in the 12-well plate, and the product of the present invention (samples 5-A, 5-B, 5-C) and the control (50 v / v% ethanol solution) were respectively added to the 12-well plate. (N = 2). The cells were cultured for 3 days under the same conditions.

  Thereafter, the culture medium of the supernatant was removed, and the cells were washed twice with PBS (2 mL / Well). Then, 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

(Confirmation of ceramide / glucosylceramide promoting effect)
First, methanol: chloroform (2.5 ml: 1.25 ml) was added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) was performed, and the supernatant a and the precipitate b were separated. The supernatant a was subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate b was subjected to the next protein amount analysis.

(1) Analysis of the amount of ceramide and the amount of glucosylceramide PBS: chloroform (1.25 ml: 1.25 ml) is added to the supernatant a, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and in the lower phase The chloroform layer was isolated and collected. The recovered chloroform layer was dried at 30 ° C. in a nitrogen atmosphere. The dried product was dissolved in 100 μl of a mixed solvent of chloroform / methanol (volume ratio = 2.5 ml: 1.25 ml) and subjected to quantification of ceramide and glucosylceramide by the following thin layer chromatography.

  A ceramide (or glucosylceramide) standard product and a sample solution were placed in a dry TLC plate, and thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. . After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide) (Cer and GlyCer, respectively, unit μg / mL).

(2) Analysis of protein amount In order to display the amount of ceramide and the amount of glucosylceramide in cells, the amount of protein was analyzed. To the precipitate b, a 900 μl SDS (sodium dodecyl sulfonate) solution was added, mixed, heated at 60 ° C. for 2 hours, and then the protein was denatured and decomposed and cooled. Subsequently, 100 μl 2N HCl was added, and the protein amount (denoted as Pro. Μg / mL) was measured by the BCA Kit method (protein amount analysis kit).

(3) Results of Analysis Cer / Pro and GlyCer / Pro were measured for each of the product of the present invention and the control sample as shown in the following formula. Table 5 shows the relative values of Cer / Pro and GlyCer / Pro according to the present invention when each of Cer / Pro and GlyCer / Pro of the control is set to 1. In addition, the relative value (Pro. (%)) Of the protein amount per well of the present invention when the amount of protein per well of the control is 100 was calculated, whereby the amount of ceramide and glucosyl per well were calculated. The amount of ceramide (relative to the control) was determined. The results are also shown in Table 5.
Cer / Well = Cer / Pro × Pro. (%)
GlyCer / Well = GlyCer / Pro × Pro. (%)

  In addition, when Cer / Pro (or Cer / Well) is higher than 1 (control), it is considered that the sample has a ceramide production promoting effect. When GlyCer / Pro (or GlyCer / Well) is higher than 1 (control), the sample is considered to have a glucosylceramide production promoting effect.

  As is clear from Table 5, the products of the present invention 5-A, 5-B, and 5-C were confirmed to have a ceramide production promoting effect.

(Example 5-2)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 5-1, Cryptococcus laurentii (No. CICIM Y0232) was transferred to Cryptococcus hungaricus (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0203). The culture was carried out in the same manner as in Example 5-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixture is used as sample 5-D of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 5-E and 5-F of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 5-E and 5-F were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 5-1, the ceramide / glucosylceramide production promoting effect of Samples 5-D, 5-E, and 5-F was evaluated. The results are shown in Table 5.

  As is clear from Table 5, it was confirmed that Samples 5-D, 5-E, and 5-F of Example 5-2 exhibited a glucosylceramide production promoting effect.

(Example 5-3)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 5-1, Cryptococcus laurentii (No. CICIM Y0232) was assigned to Cryptococcus laurentii (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0218). The culture was carried out in the same manner as in Example 5-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as sample 5-G of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as sample 5-H of the ceramide and / or glucosylceramide production promoter of the present invention, and sample H was dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. It preserve | saved in the refrigerator of 4 degreeC and used for the following cell culture.

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

  As is clear from Table 5, it was confirmed that Samples 5-G and 5-H of Example 5-3 exhibited a glucosylceramide production promoting effect.

(Example 5-4)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 5-1, Cryptococcus laurentii (No. CICIM Y0232) was assigned to Cryptococcus hungaricus (Gangnam University Industrial Microbial Resources and Information Center (CICIM as No. CICIM Y0219) The culture was carried out in the same manner as in Example 5-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as the ceramide and / or glucosylceramide production promoter 5-I of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as sample 5-J of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 5-J was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 5-1, the ceramide / glucosylceramide production promoting effect of Samples 5-I and 5-J was evaluated. The results are shown in Table 5.

  As is apparent from Table 5, it was confirmed that Samples 5-I and 5-J of Example 5-4 exhibited a glucosylceramide production promoting effect.

  For samples 5-A to 5-J, the amount of ceramide and glucosylceramide were not added to the 12-well plate containing human activated keratinocytes (ie, the cell culture was not performed). Quantity analysis was performed. Except for the above, the procedure was the same as in Example 5-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any of samples 5-A to 5-J.

  From this, ceramide or glucosylceramide does not exist in the product of the present invention itself, but the product of the present invention has an effect of promoting ceramide and / or glucosylceramide production and is used as a ceramide and / or glucosylceramide production promoter. I know you get.

(Example 6-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
Hansenula polymorpha (Purchased from Gangnam University as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0234) It was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. Furthermore, it processed for 10 minutes with the ultrasonic processing apparatus (made by Branson), and supernatant was collect | recovered by 3000 rpm and 30 mins centrifugation. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, adjusted to a concentration of 0.1% (Product 6-A-1 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 6-2)
Hansenula polymorpha (Purchased from Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, contract number CICIM Y0205) Was used in the same manner as in Example 6-1 to obtain a fermented extract of soybean residue, which was used as the ceramide and / or glucosylceramide production promoter of the present invention (product 6-B-1 of the present invention) at 4 ° C. And then subjected to the following ceramide and / or glucosylceramide production promotion test.

(Example 6-3)
Hansenula polymorpha (Purchased from Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, contract number CICIM Y0257) Was used in the same manner as in Example 6-1 to obtain a fermented extract of soybean residue, which was used as the ceramide and / or glucosylceramide production promoter of the present invention. Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 0.1 v / v% (Product 6-C-1 of the present invention), and stored at 4 ° C. And / or subjected to a glucosylceramide production promotion test.

(Example 6-4)
Hansenula polymorpha (Purchased from Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, contract number CICIM Y0122) Was used in the same manner as in Example 6-1 to obtain a fermented extract of soybean residue, which was used as the ceramide and / or glucosylceramide production promoter of the present invention (Product 6-D-1 of the present invention). Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH to 1 v / v% (present product 6-D-2) and 0.1 v / v% (present product 6-D-3). After the preparation, it is stored at 4 ° C. and subjected to the following ceramide and / or glucosylceramide production promotion test.

(Example 6-5)
Hansenula polymorpha (Purchased from Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center CICIM as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, contract number CICIM Y0135) Was used to obtain a fermented extract of soybean residue in the same manner as in Example 6-1 and used as the ceramide and / or glucosylceramide production promoter of the present invention (product 6-E-1 of the present invention) at 4 ° C. And then subjected to the following ceramide and / or glucosylceramide production promotion test.

(Example 6-6)
Hansenula ciferrii (manufactured from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0150) Was used in the same manner as in Example 6-1 to obtain a fermented extract of soybean residue, which was used as the ceramide and / or glucosylceramide production promoter of the present invention. Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1 v / v% (the product 6-F-1 of the present invention), stored at 4 ° C., and the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 6-7)
Hansenula polymorpha (Purchased from Gangnam University and commissioned by China High School Industrial Microbial Resources and Information Center CICIM as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, contract number CICIM Y0197) Was used in the same manner as in Example 6-1 to obtain a fermented extract of soybean residue, which was used as the ceramide and / or glucosylceramide production promoter of the present invention (Product 6-G-1 of the present invention). Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH to obtain 1 v / v% (invention product 6-G-2) and 0.1 v / v% (invention product 6-G-3). After the preparation, it is stored at 4 ° C. and subjected to the following ceramide and / or glucosylceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Keratinocytes culture>
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Subsequently, the medium containing no growth factor was changed in the 12-well plate, and 20 μl / well of an ethanol solution of the fermented soybean residue obtained in Examples 6-1 to 6-7 was added. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated at 60 ° C. for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. The results are also shown in Table 6.

  As shown in Table 6, the product of the present invention obtained in Example 6-2 exhibited a ceramide production promoting effect and a glucosylceramide production promoting effect. The products of the present invention obtained in Example 6-1, Example 6-3 and Example 6-6 showed a significant ceramide production promoting effect, but were obtained in Example 6-4 and Example 6-7. The product of the present invention showed a remarkable glucosylceramide production promoting effect.

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have a ceramide and / or glucosylceramide production promotion effect.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Examples 6-1 to 6-7 was analyzed only by the TLC method without performing the cell culture, and as a result, some of the products of the present invention include It was shown that ceramide / glucosylceramide was not present.

  From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

(Example 7-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
Bullera tsugae (purchased from Gangnam University as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center, CICIM Y0259) It was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. To obtain a crude extract containing the ceramide and / or glucosylceramide production promoter of the present invention (Product 7-A of the present invention).

  Further, the crude extract was treated with an ultrasonic treatment apparatus (manufactured by Branson) for 10 minutes, and the supernatant was recovered by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and adjusted to a concentration of 1 v / v% (present product 7-B) and 0.1 v / v% (present product 7-C). It preserve | saves at 4 degreeC and uses for the following ceramide and / or glucosylceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Keratinocytes culture>
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Next, the medium containing no growth factor in the 12-well plate was changed, and an ethanol solution of the fermented soybean residue obtained in Example 7-1 (Products 7-A to 7-C of the present invention) was 20 μl / Wells were added in increments. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated at 60 ° C. for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. The results are also shown in Table 7.

  As shown in Table 7, any of the products 7-A to 7-C of the present invention obtained in Example 7-1 showed a remarkable ceramide production promoting effect.

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have a ceramide and / or glucosylceramide production promotion effect.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Example 7-1 was analyzed only by the TLC method without performing the cell culture, and as a result, some of the products of the present invention include ceramide / glucosylceramide. Indicated that it does not exist.

  From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

(Example 8-1)
<Manufacture of ceramide and / or glucosylceramide production promoter (treatment of fermentation broth)>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.

  Next, 100 ml of the above medium was placed in an Erlenmeyer flask, and Rhodotorula mucilaginosa (Konan University Industrial Microbial Resources and Information Center (consigned by CICIM as number CICIM Y0250)) was inoculated in a prescribed amount and cultured at 30 ° C. for 3 days. .

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as the ceramide and / or glucosylceramide production promoter 8-A of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 8-B and 8-C of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 8-B and 8-C were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, a normal human activated keratinocyte (Cascade Co., Ltd) is used as a primary cell in a 75 cm ² culture flask (2 ml of medium containing a growth factor) in an incubator at 37 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.

  Next, the medium containing no growth factor was changed in the 12-well plate, and the product of the present invention (samples 8-A, 8-B, 8-C) and the control (50 v / v% ethanol solution) were respectively added to the 12-well plate. (N = 2). The cells were cultured for 3 days under the same conditions.

  Thereafter, the culture medium of the supernatant was removed, and the cells were washed twice with PBS (2 mL / Well). Then, 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

(Confirmation of ceramide / glucosylceramide promoting effect)
First, methanol: chloroform (2.5 ml: 1.25 ml) was added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) was performed, and the supernatant a and the precipitate b were separated. The supernatant a was subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate b was subjected to the next protein amount analysis.

(1) Analysis of the amount of ceramide and the amount of glucosylceramide PBS: chloroform (1.25 ml: 1.25 ml) is added to the supernatant a, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and in the lower phase The chloroform layer was isolated and collected. The recovered chloroform layer was dried at 30 ° C. in a nitrogen atmosphere. The dried product was dissolved in 100 μl of a mixed solvent of chloroform / methanol (volume ratio = 2.5 ml: 1.25 ml) and subjected to quantification of ceramide and glucosylceramide by the following thin layer chromatography.

  A ceramide (or glucosylceramide) standard product and a sample solution were placed in a dry TLC plate, and thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. . After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide) (Cer and GlyCer, respectively, unit μg / mL).

(2) Analysis of protein amount In order to display the amount of ceramide and the amount of glucosylceramide in cells, the amount of protein was analyzed. To the precipitate b, a 900 μl SDS (sodium dodecyl sulfonate) solution was added, mixed, heated at 60 ° C. for 2 hours, and then the protein was denatured and decomposed and cooled. Subsequently, 100 μl 2N HCl was added, and the protein amount (denoted as Pro. Μg / mL) was measured by the BCA Kit method (protein amount analysis kit).

(3) Results of Analysis Cer / Pro and GlyCer / Pro were measured for each of the product of the present invention and the control sample as shown in the following formula. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated and shown in Table 8 when each of Cer / Pro and GlyCer / Pro of the control was set to 1. In addition, the relative value (Pro. (%)) Of the protein amount per well of the present invention when the amount of protein per well of the control is 100 was calculated, whereby the amount of ceramide and glucosyl per well were calculated. The amount of ceramide (relative to the control) was determined. The results are shown in Tables 8-1 to 8-2.
Cer / Well = Cer / Pro × Pro. (%)
GlyCer / Well = GlyCer / Pro × Pro. (%)

  In addition, when Cer / Pro (or Cer / Well) is higher than 1 (control), it is considered that the sample has a ceramide production promoting effect. When GlyCer / Pro (or GlyCer / Well) is higher than 1 (control), the sample is considered to have a glucosylceramide production promoting effect.

  As is clear from Tables 8-1 to 8-2, the products of the present invention 8-A, 8-B, and 8-C were confirmed to have a ceramide production promoting effect.

(Example 2-2)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0433). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as ceramide and / or glucosylceramide production promoter 8-D of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is used as samples 8-E and 8-F of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 8-E and 8-F are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of Samples 8-D, 8-E, and 8-F was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Samples 8-E and 8-F of Example 2-2 exhibited a glucosylceramide production promoting effect.

(Example 8-3)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0430). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as a sample 8-G of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of sample 8-G was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Sample 8-G of Example 8-3 exhibited a ceramide and glucosylceramide production promoting effect.

(Example 8-4)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula glutinis (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0148). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as sample 8-H of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 8-H was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of Sample 8-H was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Sample 8-H of Example 8-4 exhibited a glucosylceramide production promoting effect.

(Example 8-5)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0324). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 8-I and 8-J of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 8-I and 8-J are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of Samples 8-I and 8-J was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Samples 8-I and 8-J of Example 8-5 exhibited a glucosylceramide production promoting effect.

(Example 8-6)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (No. CICIM Y0337). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixed solution is used as the ceramide and / or glucosylceramide production promoter sample 8-K of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of Sample K was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Sample 8-K of Example 8-6 exhibited a glucosylceramide production promoting effect.

(Example 8-7)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (CICIM as No. CICIM Y0394). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as sample 8-L of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 8-L was further dissolved in 50 v / v% EtOH to prepare a solution having a concentration of 1 v / v%. Thereafter, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of Sample 8-L was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Sample 8-L of Example 8-7 exhibited a ceramide and glucosylceramide production promoting effect.

(Example 8-8)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (No. CICIM Y0250) was assigned to Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (CICIM as No. CICIM Y0418). The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixed solution is used as a sample 8-M of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 8-N and 8-O of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 8-N and 8-O were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of samples 8-M, 8-N, and 8-O was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Samples 8-M, 8-N, and 8-O of Example 8-8 exhibited ceramide and glucosylceramide production promoting effects.

(Example 8-9)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 8-1, Rhodotorula mucilaginosa (Gangnam University Industrial Microbial Resources and Information Center (CICIM Y0250) was commissioned as Rhodotorula mucilaginosa (CICIM as CICIM Y0423) The culture was carried out in the same manner as in Example 8-1, except that the above was changed.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as sample 8-P of the ceramide and / or glucosylceramide production promoter of the present invention, and sample 8-P is further dissolved in 50 v / v% EtOH to obtain a solution having a concentration of 0.1 v / v%. After the preparation, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 8-1, the ceramide / glucosylceramide production promoting effect of Sample 8-P was evaluated. The results are shown in Tables 8-1 to 8-2.

  As is clear from Tables 8-1 to 8-2, it was confirmed that Sample 8-P of Example 8-9 exhibited a ceramide and glucosylceramide production promoting effect.

  For samples 8-A to 8-P, the amount of ceramide and glucosylceramide were not added to the 12-well plate containing the human activated keratinocytes (ie, the cell culture was not performed). Quantity analysis was performed. Except for the above, the procedure was the same as in Example 8-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any of samples 8-A to 8-P.

  From this, ceramide or glucosylceramide does not exist in the product itself of the present invention, but the product of the present invention has an effect of promoting ceramide and / or glucosylceramide production, and a ceramide and / or glucosylceramide production promoter. It can be used as

(Example 9-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
As a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, it was purchased from Sporobolomyces roseus (Gangnam University) and commissioned by China High School Industrial Microbial Resources and Information Center CICIM. Y0247) was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. Was added to obtain a crude extract of the ceramide and / or glucosylceramide production promoter of the present invention (Product 9-A of the present invention). Furthermore, it preserve | saves at 4 degreeC and uses for the following ceramide and / or glucosylceramide production promotion test.

(Example 9-2)
Sporobolomyces roseus (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0193) was used to obtain a crude extract of the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 9-1. Further, the crude extract was treated with an ultrasonic treatment apparatus (manufactured by Branson) for 10 minutes, and the supernatant was recovered by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, adjusted to a concentration of 0.1 v / v% (Product 9-B of the present invention), stored at 4 ° C., and then subjected to the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 9-3)
Sporobolomyces roseus (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0132) was used to obtain a crude extract of the ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 9-1 (Product 9-C of the present invention). Further, the crude extract was treated with an ultrasonic treatment apparatus (manufactured by Branson) for 10 minutes, and the supernatant was recovered by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1 v / v% (Product 9-D of the present invention), stored at 4 ° C., and then subjected to the following ceramide and / or glucosyl Subject to ceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Culture of keratinocytes>
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Subsequently, the medium containing no growth factor was changed in the 12-well plate, and 20 μl / well of an ethanol solution of the fermented soybean residue obtained in Examples 9-1 to 9-3 was added. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken and mixed for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated in a 60 ° C. water bath for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, mixed by shaking for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. . The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. The results are also shown in Table 9.

  As shown in Table 9, the products of the present invention obtained in Example 9-1 and Example 9-3 showed a remarkable glucosylceramide production promoting effect. The product of the present invention obtained in Example 9-2 showed a remarkable ceramide production promoting effect.

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have the effect of promoting production of ceramide and / or glucosylceramide.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Examples 9-1 to 9-3 above was analyzed only by the TLC method without performing the cell culture, and as a result, the products of the present invention include It was shown that ceramide / glucosylceramide was not present.

  From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

(Example 10-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
Brettanomyces claussenii (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, commissioned by China High School Industrial Microbial Resources and Information Center CICIM, contract number CICIM Y0125) It was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. Furthermore, it processed for 10 minutes with the ultrasonic processing apparatus (made by Branson), and supernatant was collect | recovered by 3000 rpm and 30 mins centrifugation. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 1% (product 10-A of the present invention), stored at 4 ° C., and then produced with the following ceramide and / or glucosylceramide Subject to accelerated testing.

(Example 10-2)
Brettanomyces claussenii (purchased from Gangnam University as a microorganism to produce ceramide and / or glucosylceramide production promoter from soybean residue, and commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0251) The fermented extract of soybean residue was obtained as a ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 10-1.

  Further, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, prepared to a concentration of 0.1 v / v% (the product of the present invention 10-B), stored at 4 ° C., and then the following ceramide and / or Alternatively, it is subjected to a glucosylceramide production promotion test.

(Example 10-3)
Brettanomyces claussenii (purchased from Gangnam University as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue, and commissioned by China High School Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0127) The fermented extract of soybean residue was obtained as a ceramide and / or glucosylceramide production promoter of the present invention in the same manner as in Example 10-1.

  Furthermore, the fermented extract of the soybean residue was dissolved in 50 v / v% EtOH, and adjusted to concentrations of 1 v / v% (invention product 10-C) and 0.1 v / v% (invention product 10-D), respectively. Then, it preserve | saves at 4 degreeC and uses for the following ceramide and / or glucosylceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Culture of keratinocytes>
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Subsequently, the medium containing no growth factor was changed in the 12-well plate, and 20 μl / well of an ethanol solution of the fermented soybean residue obtained in each of Examples 10-1 to 10-3 was added. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken and mixed for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated in a 60 ° C. water bath for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, mixed by shaking for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. . The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. The results are also shown in Table 10.

  As shown in Table 10, the product of the present invention obtained in Example 10-1 exhibited a ceramide production promoting effect and a glucosylceramide production promoting effect. Further, the products of the present invention obtained in Example 10-2 and Example 10-3 showed a remarkable glucosylceramide production promoting effect.

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have the effect of promoting production of ceramide and / or glucosylceramide.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Examples 10-1 to 10-3 above was analyzed only by the TLC method without performing the cell culture, and as a result, the products of the present invention include It was shown that ceramide / glucosylceramide was not present.

  From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

(Example 11-1)
(Manufacture of ceramide and / or glucosylceramide production promoter)
Yarrowia lipolytica as a microorganism for producing ceramide and / or glucosylceramide production promoter from soybean residue (purchased from Gangnam University, commissioned by China High School Industrial Microbial Resources and Information Center, CICIM Y0311) It was used.

  300 mL containing 100 mL of the ceramide / glucosylceramide production promoter production medium (10 w / v% soybean residue, 1 w / v% sucrose) obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) The inoculated flask was inoculated and cultured at 30 ° C. for 72 hours. Among them, soybean residue was prepared by putting soybean in water, impregnating it for 12 hours, allowing the soybean to sufficiently absorb water, grinding and grinding the soybean that absorbed water, filtering and drying.

  In order to purify ceramide and / or glucosylceramide production promoter from the obtained culture broth, the cells were removed by centrifugation at 3000 rpm for 10 mins, and 2.5 times the volume of anhydrous EtOH with respect to the collected culture supernatant. Furthermore, it processed for 10 minutes with the ultrasonic processing apparatus (made by Branson), and supernatant was collect | recovered by 3000 rpm and 30 mins centrifugation. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer (40 ° C. or lower) to give a fermented extract of soybean residue having a constant weight to the ceramide of the present invention. / Or glucosylceramide production promoter.

  Further, the fermented extract of the soybean residue is dissolved in 50 v / v% EtOH, adjusted to a concentration of 1%, stored at 4 ° C., and subjected to the next ceramide and / or glucosylceramide production promotion test.

(Ceramide and / or glucosylceramide production promotion test)
<Culture of keratinocytes>
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Further seeded by 2mL in 12-well Plate (number 1 × 10 ⁵ cells cells / mL), 37 ℃, it lasted cultured at 5% CO ₂ incubator until a state of 80-90% confluent.

  Subsequently, the medium containing no growth factor was changed in the 12-well plate, and 20 μl / well of an ethanol solution of the fermented soybean residue obtained in Example 11-1 was added. As a control, only ethanol (no addition of ceramide and glucosylceramide production promoter) was added. The cells were cultured for 72 hours under the same conditions. After culturing for 72 hours, the supernatant culture solution was removed, the cells were washed twice with PBS (2 mL / Well), 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for later analysis of ceramide, glucosylceramide and protein.

<Lipid extraction>
Lipid extraction was performed using the Bligh & Dyer method. Specifically, methanol: chloroform (2.5 mL: 1.25 mL) was first added to the PBS solution containing the collected cells and stirred, and then shaken and mixed for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) is performed to separate the supernatant A and the precipitate B, and the supernatant A is subjected to ceramide amount and glucosylceramide amount analysis, and the precipitate B is subjected to the next protein amount analysis.

<Protein content analysis>
Protein amount analysis was performed to display the amount of ceramide and glucosylceramide in the cells. The recovered precipitate B was mixed with 900 μl SDS solution, heated in a 60 ° C. water bath for 2 hours, and then cooled to denature and decompose the protein. Subsequently, 100 μl 2N HCl was added, and the amount of protein (μg / mL) was measured by the BCA Kit method.

<Analysis of ceramide amount and glucosylceramide amount>
PBS: chloroform (1.25 mL: 1.25 mL) was added to the remaining supernatant A, mixed by shaking for 20 minutes, centrifuged (3000 rpm, 5 minutes), and the chloroform layer in the lower phase was isolated and recovered. . The recovered chloroform layer was dried at 30 ° C. under nitrogen reflux to obtain a dried product. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml), and ceramide and glucosylceramide were quantified by the following thin layer chromatography (respectively referred to as Cer and GlyCer, unit μg / mL) )

  Using a dry TLC plate, thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. The phosphoric acid solution of copper sulfate was sprayed and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide).

  Then, from the following formula 1, the obtained value is divided by the amount of each protein, and Cer / Pro and GlyCer / Pro are obtained. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated by setting Cer / Pro and GlyCer / Pro of the control to 1, respectively. The amount of protein per well of the control was defined as 100, and the amount of ceramide and the amount of glucosylceramide per well (respectively referred to as Cer / Well and GlyCer / Well, relative amounts to the control) were determined. The results are also shown in Table 11.

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

  Therefore, the ceramide and / or glucosylceramide production promoter of the present invention was confirmed to have the effect of promoting production of ceramide and / or glucosylceramide.

(Test to prove that ceramide and / or glucosylceramide is not present in ceramide and / or glucosylceramide production promoter)
The ceramide and / or glucosylceramide production promoter obtained in Example 11-1 was analyzed only by the TLC method without performing the cell culture, and as a result, some of the products of the present invention include ceramide / glucosylceramide. Indicated that it does not exist.

  From this, it is confirmed that the product of the present invention does not contain ceramide or glucosylceramide, but has an effect of promoting ceramide and / or glucosylceramide production, and can be used as a ceramide and / or glucosylceramide production promoter. It was done.

Claims (36)

  A method for producing a ceramide and / or glucosylceramide production promoter, wherein yeast is cultured in a medium containing soybean residue and saccharide, and a ceramide and / or glucosylceramide production promoter is obtained from the culture supernatant.   The yeast is Schizosaccharomyces yeast, Pichia yeast, Candida yeast, Debaryomyces yeast, Cryptococcus yeast, Hansenula yeast, Bullera yeast, Rhodotorula yeast, Sporobolomyces yeast, Brettanomyces yeast, The method for producing a ceramide and / or glucosylceramide production promoter according to claim 1, wherein the ceramide and / or glucosylceramide production promoter is at least one kind selected from yeasts of the genus and yeasts of the genus Yarrowia.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast belonging to the genus Schizosaccharomyces is Schizosaccharomyces octoporus.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Pichia is Pichia anomala, Pichia guilliermondii, Pichia norvegensis, Pichia membranifaciens or Pichia burtonii.   The yeast of the genus Candida is Candida zeylanoides, Candida boidinii, Candida parapsilosis, Candida magnoliae, Candida. -Candida maltosa, Candida steatolytica, Candida butyri, Candida rhagii or Candida metapsilosis Of producing a ceramide and / or glucosylceramide production promoter.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast belonging to the genus Debaryomyces is Debaryomyces hansenii or Debaryomyces vanrijiae.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Cryptococcus is Cryptococcus laurentii or Cryptococcus hungaricus.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Hansenula is Hansenula polymorpha or Hansenula ciferrii.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Bullera is Bullera tsugae.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast belonging to the genus Rhodotorula is Rhodotorula mucilaginosa or Rhodotorula glutinis.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Sporobolomyces is Sporobolomyces roseus.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Brettanomyces is Brettanomyces claussenii.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 2, wherein the yeast of the genus Yarrowia is Yarrowia lipolytica.   The method for producing a ceramide and / or glucosylceramide production promoter according to any one of claims 1 to 13, wherein the saccharide is sucrose.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 14, wherein the medium comprises soybean residue, sucrose and water.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 15, wherein the soybean residue is contained in the medium at 8 to 12 w / v% and the saccharide at 0.8 to 1.2 w / v%. .   The method for producing a ceramide and / or glucosylceramide production promoter according to any one of claims 1 to 16, wherein the culture temperature is 27 to 35 ° C.   The method for producing a ceramide and / or glucosylceramide 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 glucosylceramide production promoter according to any one of claims 1 to 18, wherein the supernatant is obtained by centrifuging the culture solution obtained by the culture.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 19, wherein ethanol is added to the centrifuged supernatant to obtain a ceramide and / or glucosylceramide production promoter.   21. The ceramide and / or glucosyl according to claim 20, wherein the ethanol added is further purified by ultrasonic treatment, centrifugation, filtration, and vacuum drying to obtain a ceramide and / or glucosylceramide production promoter. A method for producing a ceramide production promoter.   Use of a fermented extract of soybean residue obtained by culturing yeast in a medium containing soybean residue and saccharides for promoting ceramide and / or glucosylceramide production.   Use of a fermented extract of soybean residue obtained by culturing yeast in a medium containing soybean residue and sugar as a ceramide and / or glucosylceramide production promoter.   Use of a fermented extract of soybean residue obtained by culturing yeast in a medium containing soybean residue and sugar for the production of a ceramide and / or glucosylceramide production promoter.   The yeast is Schizosaccharomyces yeast, Pichia yeast, Candida yeast, Debaryomyces yeast, Cryptococcus yeast, Hansenula yeast, Bullera yeast, Rhodotorula yeast, Sporobolomyces yeast, Brettanomyces yeast, The use according to any one of claims 22 to 24, which is at least one kind of yeast selected from yeast of the genus and yeast of the genus Yarrowia.   26. Use according to claim 25, wherein the yeast of the genus Schizosaccharomyces is Schizosaccharomyces octoporus.   26. Use according to claim 25, wherein the yeast of the genus Pichia is Pichia anomala, Pichia guilliermondii, Pichia norvegensis, Pichia membranifaciens or Pichia burtonii.   The yeast of the genus Candida is Candida zeylanoides, Candida boidinii, Candida parapsilosis, Candida magnoliae, Candida. 26. The claim according to claim 25, which is Candida maltosa, Candida steatolytica, Candida butyri, Candida rhagii or Candida metapsilosis. Use of.   26. Use according to claim 25, wherein the yeast of the genus Debaryomyces is Debaryomyces hansenii or Debaryomyces vanrijiae.   26. Use according to claim 25, wherein the yeast of the genus Cryptococcus is Cryptococcus laurentii or Cryptococcus hungaricus.   26. Use according to claim 25, wherein the yeast of the genus Hansenula is Hansenula polymorpha or Hansenula ciferrii.   26. Use according to claim 25, wherein the yeast of the genus Bullera is Bullera tsugae.   26. Use according to claim 25, wherein the yeast of the genus Rhodotorula is Rhodotorula mucilaginosa or Rhodotorula glutinis.   26. Use according to claim 25, wherein the yeast of the genus Sporobolomyces is Sporobolomyces roseus.   26. Use according to claim 25, wherein the yeast of the genus Brettanomyces is Brettanomyces claussenii. 26. Use according to claim 25, wherein the yeast of the genus Yarrowia is Yarrowia lipolytica.