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WO2012062043A1 - Procédé de production d'un promoteur à partir de céramide et/ou de glucosylcéramide - Google Patents

Procédé de production d'un promoteur à partir de céramide et/ou de glucosylcéramide Download PDF

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Publication number
WO2012062043A1
WO2012062043A1 PCT/CN2011/001877 CN2011001877W WO2012062043A1 WO 2012062043 A1 WO2012062043 A1 WO 2012062043A1 CN 2011001877 W CN2011001877 W CN 2011001877W WO 2012062043 A1 WO2012062043 A1 WO 2012062043A1
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Prior art keywords
ceramide
yeast
glucosylceramide
production promoter
glucose
Prior art date
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PCT/CN2011/001877
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English (en)
Chinese (zh)
Inventor
孔凡旗
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Kao Corp
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Kao Corp
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Filing date
Publication date
Priority claimed from CN201110289106.4A external-priority patent/CN102465151B/zh
Priority claimed from CN201110289078.6A external-priority patent/CN102994557B/zh
Priority claimed from CN201110289092.6A external-priority patent/CN102477446B/zh
Priority claimed from CN201110289008.0A external-priority patent/CN102994555B/zh
Priority claimed from CN201110289055.5A external-priority patent/CN102465150B/zh
Priority claimed from CN201110289054.0A external-priority patent/CN102994556B/zh
Priority claimed from CN201110289082.2A external-priority patent/CN102994558B/zh
Priority claimed from CN201110316466.9A external-priority patent/CN103060382B/zh
Priority claimed from CN201110316486.6A external-priority patent/CN103060383B/zh
Priority claimed from CN201110317308.5A external-priority patent/CN103060385B/zh
Priority claimed from CN201110316497.4A external-priority patent/CN103060384B/zh
Priority to JP2013522070A priority Critical patent/JP5639713B2/ja
Application filed by Kao Corp filed Critical Kao Corp
Publication of WO2012062043A1 publication Critical patent/WO2012062043A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

Definitions

  • the present invention relates to a method for producing a neuronal amide production promoting agent and/or a glucosylceramide production promoting agent capable of promoting skin production of ceramide and/or glucosylceramide. More particularly, the present invention relates to the production of a ceramide and/or glucosylceramide production promoter capable of promoting skin production of ceramide and/or glucosylceramide by using a specific microorganism, a by-product okara of a food processing industry or the like as a main raw material of a culture medium. method. Background technique
  • the skin has the ability to produce ceramide and glucosylceramide.
  • Ceramide synthesized by epidermal cells is secreted in the skin, especially in the cuticle of the epidermis, and is a major component of intercellular lipids. Ceramide, one of the sphingolipids, is present in the stratum corneum of the skin and accounts for about 50% of the intercellular lipids in the stratum corneum.
  • Sphingolipid is a generic term for a complex lipid having a sphingosine skeleton.
  • Glucosylceramide is a sphingolipid that combines glucose with ceramide.
  • the ceramide synthesized by epidermal cells is temporarily stored in the form of glucosylceramide or sphingomyelin; it is then excreted outside the cell, in glucocerebrosidase and neurophospholipidase (sphingomyelinase). Under the action of ceramide, ceramide is formed again, thereby functioning as an intercellular lipid. Ceramide and glucosylceramide in the skin have physiological effects such as moisturizing function of the skin and barrier function of the skin.
  • Patent Documents 1 to 2 It has been known that sphingolipids such as ceramide, glucosylceramide, and galactosylceramide have effects of promoting the retention of water in the stratum corneum and improving skin roughness (Patent Documents 1 to 2). Although the skin has the ability to produce ceramide and glucosylceramide, the skin tends to become dry due to environmental, climatic, and the like. Therefore, it is not sufficient to produce ceramide by the skin itself to improve the moisturizing function of the skin.
  • Patent Document 3 describes a method for producing glucosylceramide by culturing Candida tropicalis (Owifefe tropicalis) JPCCY0004 strain (NITE P-570) by using Candida (0«3 ⁇ 43 ⁇ 4) It can be applied to the dry skin of the human body to improve the skin. Skin moisturizing function.
  • this method is not a method for ceramide and/or glucosylceramide production promoter, but merely obtains a large amount of glucosylceramide present directly from the cultured cells, and thus the nerve of the synthesized glucosylceramide
  • the amide skeleton is different from the ceramide skeleton of ceramides present in the human body, and has carbon-carbon double bonds at two places and has a branch. Therefore, when such glucosylceramide directly extracted from microorganisms is applied to human skin, there is a possibility that it cannot be adapted to human skin and causes problems in use safety.
  • the method of supplementing ceramide to the skin from the outside has a problem that the moisturizing effect is insufficient in persistence as in the conventionally used moisturizing agent, and the skin in some cases is insufficiently absorbed by the externally supplied ceramide or the like, thereby failing to be performed. Give full play to your moisturizing effect.
  • Patent Document 7 discloses a ceramide synthesis promoter capable of activating ceramide synthesis of epidermal cells themselves in the surface layer of the skin, which comprises a bacterium culture containing nicotinic acid and/or nicotinamide as an active ingredient.
  • the culture of the bacterium may be a lactic acid bacteria culture, a bifidobacterium culture, a lentinus bacillus culture or a yeast culture.
  • the ceramide production amount of the lactic acid bacteria culture or the yeast culture (Comparative Example ⁇ 4) containing no nicotinic acid and/or nicotinamide was not significantly changed from the control group.
  • the ceramide synthesis promoter substantially contains nicotinic acid and/or nicotinamide as an active ingredient, and the effect of promoting ceramide synthesis is not sufficient, and there is still room for improvement.
  • the medium used in the preparation of the accelerator substantially contains nicotinic acid and/or nicotinamide as an active ingredient, the preparation cost of the ceramide synthesis accelerator is high.
  • yeast is a single-celled fungus that is widely distributed in nature and is mainly grown in a slightly acidic, sugary environment. Can survive in an oxygen-deficient environment. Can be used for brewing production.
  • yeasts including yeasts of the genus iSchizosaccharomyces, yeast of the genus Pichia, Candida, yeast of Debaryomyces, and iCryptococcus.
  • Schizosaccharomyces Schi noodles ccharomyces yeast has long been used in food processing, which produces ethanol and is also used as a research material for biology.
  • Pichia genus yeast has the following properties, uses, and the like.
  • Pichia cell lines are spherical, elliptical, elongated, occasionally conical, but do not form a apex.
  • Asexual reproduction is a multilateral budding, and some species can form spores.
  • Each ascus usually contains 1 to 4 ascospores, occasionally more than 4.
  • the spores are cap-shaped, hemispherical or spherical.
  • Both hyphae and pseudohyphae can be used as ascospores, but the morphology does not become large or become spindle-shaped, with the same or different combinations; can be assimilated nitrate, DBB reaction is negative.
  • Pichia The taxonomic status of Pichia is: Ascomycetes (o? > ⁇ to), Semi-Ascomycetes ⁇ Hemiascomycetes, Saccharomycetales, Saccharomycetaceae, Pichia (Combies czz') o).
  • Pichia iPichia is, for example, the use of methanol to produce useful substances such as antibiotics, biological proteins, etc.; or it can also be used as genetically engineered bacteria.
  • Pichia pastoris is used to produce reduced glutathione, and other Pichia pastoris are used to produce some proteins/enzymes.
  • Candida Ccmdick's microorganisms usually have the following uses: for example, Candida crudea (Candida valida is used to produce lipase, Candida tropicalis Candidametapsilosis, Candida utilis (cmdida utilis, etc.) produce Brewing, glycerin, edible yeast, organic acids, and enzyme preparations can also be used in the word industry; and, the microorganisms of Candida Ccmdida can also be used to treat waste from industrial and agricultural by-product processing industries, and to produce edible proteins.
  • the microorganism of Debaryomyces generally has the following industrial uses, for example, fermenting glucose to produce D-arabitol, or converting xylose into xylitol.
  • microorganisms of the genus Hansenula ficm la are capable of producing ethyl acetate, thereby increasing the flavor of the product, and thus are generally used in the wine making and food industries.
  • microorganisms of the genus Bulrush, Bi le are generally used for food fermentation, such as sausage fermentation.
  • Rhodotorula In addition, some studies have been conducted on the microorganisms of Rhodotorula. For example, certain species of the genus Rhodotorula have weak oxidation of hydrocarbons and can synthesize beta-carotene. For example, the sticky red yeast variety can oxidize terpene to produce fat, and the content can reach 50 ⁇ 60% of dry biomass. Under certain conditions, a-alanine and glutamic acid can also be produced, and the ability to produce methionine is also strong, up to 1% of dry biomass.
  • microorganisms of the sporozoite sporobolomyces are useful in industries such as accumulation of coenzyme Q10 in bacteria.
  • the microbes of Sporobo myces are also attracting attention as fungi for carotenoids, L-carnitine and other useful substances.
  • the microorganism of the genus Brettanomyces belongs to yeast, which is widely found in nature and usually multiplies in barrel storage to produce ethyl phenol. Therefore, in the wine industry, it is generally unpopular as a contaminating bacteria that destroys the aroma of beer, wine, and the like.
  • Yarrowia a microorganism such as rra 'a lipolytica, is known to be particularly useful for the production of polyunsaturated fatty acids (PUFAs).
  • PUFAs polyunsaturated fatty acids
  • Patent Document 1 Japanese Patent Laid-Open No. 61-260008
  • Patent Document 2 Japanese Unexamined Patent Publication No. 61-271205
  • Patent Document 3 JP-A-2010-22217
  • Patent Document 4 Japanese Special Report No. 2005-194240
  • Patent Document 6 Japanese Special Feature No. 2010-150237
  • Patent Document 7 Japanese Patent Publication No. 9-194383 SUMMARY OF THE INVENTION
  • the present invention has been made to solve the above-mentioned problems in the prior art, and an object thereof is to provide a method for producing a ceramide production promoter and/or a glucosylceramide production promoter at low cost and high efficiency.
  • the present inventors conducted intensive studies to solve the above-described technical problems, and as a result, found that: culturing yeast in a medium containing okara and sugar can obtain a ceramide production promoter and/or a glucosylceramide production promoter at low cost and high efficiency.
  • the invention has thus been completed.
  • the present invention provides a method for producing a ceramide production promoter and/or a glucosylceramide production promoter, wherein a yeast is cultured in a medium containing okara and a saccharide, and a ceramide production promoter is obtained from the supernatant thereof. And / or glucosylceramide production enhancer.
  • the present invention also relates to the use of a fermented extract of bean dregs for promoting the production of ceramide and/or glucose amide, which is obtained by culturing yeast in a medium containing okara.
  • the present invention also relates to the use of a fermented extract of bean dregs as a ceramide production promoter and/or a glucosylceramide production promoter, which is obtained by culturing yeast in a medium containing soybean dregs.
  • the present invention also relates to the use of a fermented extract of bean dregs for producing a ceramide production promoter and/or a glucosylceramide production promoter, wherein the fermented extract of the bean dregs is obtained by culturing yeast in a medium containing okara.
  • the yeast may be selected from the group consisting of the yeast of the genus Chinosaccharomyces, the yeast of the genus iPichia, the yeast of Candida ⁇ Can Canida, the yeast of Debaryomyces, the yeast of Cryptococcus i. Saccharomyces (Hans Let La yeast, B. ssp. Bid ra) Yeast, Rhodotorula yeast, Saccharomyces sp. iSporobolomyces yeast, Brettmomyces yeast, Yarrow (rraw) ⁇ ) At least any one of the yeasts.
  • a specific microorganism can be used and a readily available substance such as bean dregs can be used as a main raw material of the medium, whereby a ceramide production promoter and/or a glucosylceramide production promoter can be obtained inexpensively and efficiently.
  • the accelerator can effectively improve the skin's ability to produce ceramide/glucose ceramide, thereby effectively improving the skin's moisturizing ability.
  • yeast is cultured in a medium containing okara and saccharides, and a ceramide production promoter and/or a glucosylceramide production promoter is obtained from the supernatant.
  • the microorganism which can be used in the production of the ceramide and/or glucosylceramide production promoter of the present invention may be selected from the group consisting of Schizosaccharomyces yeast, Pichia yeast, Candida iCandidO yeast, Debali. Saccharomyces cerevisiae (Debaryomyces yeast, Cryptocaryon genus (Cr_y;?
  • Examples of the microorganism of the Schizophyllum haromyces which can be used in the present invention include Schizosaccharomyces octo?on «, Schizosaccharomyces pombe, and the like which are commonly used in the food industry. Also included are variants of these microorganisms obtained according to the usual methods.
  • Schizosaccharomyces octoporus is preferred from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting effect.
  • Examples of the microorganism of the Pichia genus Picic0 which can be used in the present invention include the Pichia pastoris Pichi, which is commonly used in the food industry, and Pichia guilliermondii 5
  • Pichia anomala is preferred, Pichia guilliermondii, Norwegian Bi Pichia norvegensis ⁇ Pichia membranifaciens Pichia burtonii.
  • Pichia membranifaciens is also known as Pichia membranaefaciens, Pi Pilot alcoholophila,
  • Pichia burtonii Also known as Candida (Candida) Dematium chodati, Endomycopsis chodati (Hyphopichia burtonii) or T. chosporon behrendi.
  • Candida parapsilosis Ccmdida magnoliae
  • Candida maltosa Candida steatolyticcd
  • Candida butyri Candida rhagi
  • microorganisms function as follows, and by culturing in a medium containing okara and saccharides, ceramide and/or glucosylceramide which enhance epidermal cells can be obtained from the supernatant thereof. a substance that produces capacity.
  • the product obtained has a higher ceramide
  • Candida zeylanoides Candida boidinii, Candida parapsilosis, Magnolia pseudoacacia Candida magnoliae, Candida maltosa, Candida steatolytica (synonym/previous name: Candida hellenical), Candida butyr (Candida butyr , Candida rhagii or Candida metapsilosis.
  • Examples of the microorganism of the genus Debcuyomyces which can be used in the present invention include Debaryomyces hameniO and Deb ⁇ co/jwo ⁇ which are used from the food field. /n ⁇ At least one microorganism selected from the yeast Debaryomyces vanrijiae, etc.; and a mutant strain of these microorganisms obtained according to a usual method.
  • microorganisms function as follows, by culturing in the presence of okara and sugar The medium is cultured, whereby a substance for increasing the ceramide and/or glucosylceramide production ability of the epidermal cells can be obtained from the supernatant thereof, wherein the obtained product has a higher ceramide production promoting effect and/or glucose.
  • ceramide production promoting effect viewpoint it is preferable Debaryomyces hansenii (Debaryomyces hansenii), or yeast Fanlideba Wo 1 J (Debaryomyces vanrijiae)
  • microorganism of the cryptococcal yptococcus which can be used in the present invention include Cryptococcus hungaric sinensis, Cryptococcus hungaric s, Cryptococcus terreus, and the like; These include mutant strains of these microorganisms obtained according to the usual methods.
  • the product obtained from the viewpoint of having higher ceramide production promoting effect and / or glucose ceramide production promoting effect preferably Cryptococcus laurentii (Cryptococcus laurentii) 1 J Wo Hungary or Cryptococcus (Cryptococcus Hungaricus).
  • microorganism of the genus Hansenula which can be used in the present invention include Ham ula polymorpha and Hansenula sinensis (Ha Song mda ciferrii, sub-membrane Han) used from the food field. "Shen Yeast Hansenu subpelliculosa" ⁇ at least one microorganism selected from Hansenu satumus (iHansenula anomala), etc.; Method for obtaining mutant strains of these microorganisms.
  • microorganisms function as follows, and by culturing in a medium containing okara and saccharides, a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant.
  • a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant.
  • Hansenula polymorpha or Schwannella Ha-resolved La cifetrif the homologous/previous name of Hansenula polymorpha): Pichia angusta, Hansemda angusta, polymorpha Ogataea polymorpha ).
  • the spruce-sucking yeast Bt m tsugae used in the food field can be cited.
  • Polygonum buckwheat (Bu!lera Cao iabilis) At least one microorganism selected from the genus Bullera albus, etc.; and a mutant strain of these microorganisms obtained according to a usual method.
  • microorganisms function as follows, in a medium containing okara and sugar By culturing, a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant thereof, wherein the obtained product has a higher ceramide production promoting effect and/or glucosylceramide. From the viewpoint of producing a promoting effect, Spirogymus sinensis iBulkm tsugae (synonym/previous name: Sporobolomyces tsugae) is preferred.
  • Rhodotorchus Rhocto examples include Rhodotola mucilaginosa, Rhodotorula glutinis, Rhodotorula minuta, and Rhodotorula pallida. , Rhodotorula rubra, etc.; also includes variants of these microorganisms obtained according to the usual methods.
  • Rhodotorula mucilaginosa or Rhodotorula glutinis is preferred from the viewpoint that the obtained product has a higher ceramide production promoting effect and/or a glucose ceramide production promoting effect.
  • the microorganism of hmy CeS may, for example, list at least one microorganism selected from the group consisting of Spowbohmyces roseus, the genus Sporobolomyces salmonicolor, or the porphyry sporobolomyces ruber; According to A variant of these microorganisms obtained by the usual method.
  • These microorganisms function as follows, and by culturing in a medium containing okara and saccharides, a substance capable of increasing ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant. Among them, from the viewpoint that the obtained product has a higher ceramide production-promoting effect and/or a glucosylceramide-promoting effect, Sporozolios roseus is preferred.
  • the microorganism of the genus Brett momyces which can be used in the present invention is selected from the group consisting of Brettanomyces claussenii or Brettanomyces anomalus used in the food field. At least one microorganism; and a variant of these microorganisms obtained according to a usual method. These microorganisms function as follows, and are cultured in a medium containing okara and saccharides, whereby substances capable of increasing ceramide and/or glucosylamide production ability of epidermal cells can be obtained from the supernatant. Among them, Brettanomyces claussenii is preferred from the viewpoint that the obtained product has a higher ceramide production promoting effect and/or a glucosylceramide production promoting effect.
  • microorganisms include yeast Yarrow lipolytica (Y ar that ia lipolytic ⁇ or the like according to conventional method yeast Yarrow lipolytica obtained (Yarn ia lipofytica Such mutants of microorganisms. These microorganisms function as follows, and by culturing in a medium containing okara and saccharides, ceramide and/or glucosylceramide production ability of epidermal cells can be obtained from the supernatant thereof.
  • Yarrowia lipolytic ⁇ is preferred, and its synonym/has The names are: Caw3 ⁇ 4 ⁇ 4 ⁇ 4 lipolytica, (Candida paralipolytica), Saccharomycopsis lipolytica., Endomycopsis lipolytica, Candida albicans (Pseudomonilia deformans).
  • the microorganisms (yeast) to which the present invention relates can be purchased from commercial sources such as the China General Microorganisms Collection and Management Center (CGMCC), the China Center for Type Culture Collection (CCTCC), and the Industrial Microbial Resources and Information Center (CICIM) of Jiangnan University.
  • CGMCC China General Microorganisms Collection and Management Center
  • CTCC China Center for Type Culture Collection
  • CICIM Industrial Microbial Resources and Information Center
  • one type of the above microorganism may be used alone, or two or more types of the above microorganisms (yeast) may be used in combination.
  • Method for producing ceramide and/or glucosylceramide production promoter comprises culturing yeast in a medium containing okara and saccharides, and A ceramide and/or glucosylceramide production promoter is obtained in the supernatant. The specific steps of the method are as follows.
  • the medium used in the present invention is a medium containing bean dregs and sugar as a main component. Both the okara and the saccharide are easily available, and thus, the medium of the present invention has an advantage of being low in cost. Further, in addition to this, for example, 1% peptone, 0.5% yeast powder or the like may be added as appropriate.
  • the bean dregs refers to a substance obtained by immersing soybeans in water to sufficiently absorb water, pulverizing, filtering, removing water, and draining. It is also possible to use soybean residue remaining in the soybean product manufacturing process such as pressing soybean milk in the food industry.
  • the saccharide may be glucose, sucrose or maltose, etc., and they may be used singly or in combination. Further, from the viewpoints of improving the productivity and utilization of the fermentation, and the effect of promoting the ceramide production promoting effect and the glucosylceramide production promoting effect, glucose is preferred.
  • the medium of the present invention is preferably composed of okara, glucose, and water from the viewpoint of enhancing the ceramide production-promoting effect and the glucosylceramide production-promoting effect, and more preferably the content of the bean dregs in the medium is 5 to 20 w/v%, more preferably 8 to 12 w/v%, the content of glucose is 0.2 to 2 w/v%, more preferably 0.8 to 1.2 w/v%.
  • the cells of the above microorganisms are directly inoculated into a ceramide/glucose ceramide production promoter production medium.
  • a culture condition for producing a ceramide and/or a glucosylceramide production promoter the physiological characteristics of the yeast microorganism, the temperature and time necessary for optimizing the growth of the yeast microorganism, and the ceramide production promoting effect and/or glucose
  • it is preferably cultured at a culture temperature of 20 to 40 ° C, more preferably 27 to 35 ° C for 1 to 7 days, preferably for 48 to 96 hours, more preferably for 68 to 76 hours.
  • the purification method can be carried out by a combination of a usual operation such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, crystallization, or the like as needed.
  • the supernatant is preferably obtained by centrifugation.
  • the speed of the centrifugation is preferably from 2000 to 4000 rpm, more preferably from 2500 to 3500 rpm, and the centrifugation time is preferably from 0 to 20 minutes, more preferably from 5 to 15 minutes.
  • ethanol may be added to the above supernatant obtained after centrifugation for sterilization, and the supernatant to which ethanol is added may be used as a ceramide production promoter and/or a glucosylceramide production promoter.
  • the cells can also be removed from the culture solution by centrifugation, ultrasonically treated, centrifuged, and the supernatant is recovered. Next, the recovered supernatant is filtered to remove impurities and the like, and dried under vacuum to obtain a ceramide and/or glucosylceramide production-promoting agent of the present invention.
  • the soybean residue fermentation extract obtained by culturing the yeast of the present invention and obtained from the culture supernatant has an effect of increasing the amount of ceramide and/or glucosylceramide in keratinocytes of normal humans.
  • the soybean residue fermentation extract obtained by cultivating the yeast of the present invention and obtained from the culture supernatant can be used for promoting therapeutic or non-therapeutic purposes of ceramide and/or glucosylceramide production, and can also be used as a treatment.
  • Use for non-therapeutic purposes is for cosmetic purposes, or for maintaining a healthy state of use.
  • the use of the therapeutic purpose and the use of non-therapeutic purposes can be performed in a completely differentiated manner.
  • the soybean residue fermentation extract obtained by culturing the yeast of the present invention and obtained from the culture supernatant can also be used for producing a ceramide production promoter and/or a glucosylceramide production promoter.
  • the ceramide production promoting agent and/or the glucosylceramide production promoting agent of the present invention can be used as a pharmaceutical or quasi-medicine for increasing and recovering ceramide and/or glucosylceramide in the stratum corneum and improving the barrier function and moisturizing function of the skin.
  • the ceramide production promoter or glucosylceramide production promoter can also be used as The use of quasi-drugs and cosmetics, which is based on the concept of ceramide production promotion or promotion of glucosylceramide production, and which indicates the concept as needed.
  • the form of use of the ceramide production promoter and/or the glucosylceramide production promoter of the present invention may be an additive for addition to cultured cells, or may be incorporated into a skin external preparation such as a skin cleansing agent or a cosmetic.
  • a skin external preparation such as a skin cleansing agent or a cosmetic.
  • various forms such as a lotion, an emulsion, a gel, a cream, an ointment, a powder, and a granule may be mentioned.
  • the base as the external preparation is not particularly limited as long as it is a known external base.
  • the ceramide production promoting agent and/or the glucosylceramide production promoting agent of the present invention or the oily component usually blended with the skin external preparations may be used singly.
  • the amount of the dried product as the accelerator is preferably added. It is 0.0001 to 10 w/v%, more preferably 0.001 to 5 w/v%. When the addition amount is 0.0001 w/v% or more, sufficient promotion effect can be obtained; and when the addition amount is 10% or less, the irritation to epidermal cells is smaller.
  • the blending amount is preferably from 0.01 to 20 w/v%, more preferably from 0.1 to 10 w/v%, based on the total amount of the external preparation for skin, from the viewpoint of the color and odor of the culture.
  • the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. Broken, filter it The liquid component is removed, and the remaining residue is drained to obtain okara.
  • the okara was mixed with glucose and water, and the concentrations of the okara and glucose in the mixture were respectively 10 w/v ° / ⁇ B lw / v%, and the mixture was used as a medium.
  • the resulting culture was then centrifuged to GOOO rpm for 10 minutes).
  • the remaining supernatant was diluted with 2.5 times absolute ethanol, and the diluted ethanol mixture was sonicated for 10 minutes, centrifuged at 80 rpm for 30 minutes, and the supernatant (about 70 ml) was collected.
  • the supernatant was filtered through a filter paper.
  • the filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer, and the dried product was used as the sample 1 - A, 1 - B of the ceramide and/or glucosylceramide production promoter of the present invention, and the sample 1 - A, 1 was used.
  • Each of -B was dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%, and stored in a refrigerator at 4 ° C as an additive for cell culture.
  • Cell culture normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at 37 ° C, C0 2 concentration. The cells were cultured to a concentration of 80 to 90% under conditions of 5%. After passage, the cells were passaged into a 175 cm 2 square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per cell at a cell concentration of 1 ⁇ 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • CHCB CH30H: CH3COOH (190: 9: 1 v/v/v) as a layering agent.
  • the layer is applied to the top of the sheet and the spreader is blown dry with a blower; the above steps are repeated once.
  • CHCB: CH30H: C3H60 76: 20: 4 v/v/v
  • the layering agent wait for the layering agent to travel 2.5 cm from the bottom of the thin plate, stop the layer, and blow the hair with a hair dryer.
  • inventive products 1-A and 1-B have a ceramide production promoting effect and/or a glucosylceramide production promoting effect.
  • the ceramide/glucose ceramide amount was directly analyzed without adding it to the above-mentioned 12-well plate containing keratinocytes (that is, without performing the above cell culture).
  • the other operations were carried out in the same manner as in the above Example 1-1. As a result, it was confirmed that no ceramide or glucosylceramide was present in the samples 1-A to 1-B themselves.
  • the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. It is crushed, filtered and the liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentrations of the okara and glucose in the mixture were respectively 10 w/ 3 ⁇ 4 ⁇ n lw/v%, and the mixture was used as a medium.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucosylceramide-producing agent-producing sample 2-A of the present invention, and stored in a refrigerator at 4 ° C for use in cell culture. Additives.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at 37 ° C and a CO 2 concentration of 5%. The cells were cultured to a concentration of 80 90%. After passage, the cells were passaged into a 175 cm 2 square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per well, and the cell concentration was IX 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • the ceramide (or glucosylceramide) standard and sample solution are spotted onto a dry TLC plate and chromatographed with CHC13 : CH30H: CH3COOH (190: 9: 1 v/v/v) as a layering agent.
  • the layer is applied to the top of the sheet, and the layer spreader is blown by a blower; the above steps are repeated once.
  • CHCB: CH30H: C3H60 76: 20: 4 v/v/v
  • Example 2-2 As shown in Table 2, it was confirmed that the product 2-A of the present invention has a ceramide production promoting effect and a glucosylceramide production promoting effect.
  • Example 2-2 As shown in Table 2, it was confirmed that the product 2-A of the present invention has a ceramide production promoting effect and a glucosylceramide production promoting effect.
  • Example 2-1 Pichia norvegensis (CICIM, numbered CICIM Y015 1 deposited at the Jiangnan University Industrial Microbial Resources and Information Center) was used instead of the abnormality. In the same manner as in Example 2-1, except that Pichia anomala was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the sample 2-D of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use in cell culture. Additives.
  • Samples 2-E and 2-F which are ceramide/glucosylceramide production promoters of the present invention, were prepared by dissolving the samples 2-E and 2-F in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture.
  • the ceramide/glucose ceramide production promoting effect of the samples 2-D, 2-E, and 2-F was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2.
  • Example 2-4 As shown in Table 2, it was confirmed that the samples 2-D, 2-E, and 2-F of Example 2-3 had a ceramide-promoting effect.
  • Example 2-4 As shown in Table 2, it was confirmed that the samples 2-D, 2-E, and 2-F of Example 2-3 had a ceramide-promoting effect.
  • Example 2-4 As shown in Table 2, it was confirmed that the samples 2-D, 2-E, and 2-F of Example 2-3 had a ceramide-promoting effect.
  • Example 2-4 As shown in Table 2, it was confirmed that the samples 2-D, 2-E, and 2-F of Example 2-3 had a ceramide-promoting effect.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the Pichia pastoris (a Pichi discussion! a (preserved at the Industrial Microbial Resources and Information Center of Jiangnan University, CICIM, No. CICIM Y0297) was used instead. The culture was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 2-G of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • the supernatant (about 70 ml) was collected, the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight as a sample of the ceramide/glucose ceramide-producing agent of the present invention.
  • 2-H the sample 2-H was dissolved in 50% ethanol to prepare a 1% strength solution, and stored in a refrigerator at 4 °C as an additive for cell culture.
  • Example 2-5 As shown in Table 2, it was confirmed that the samples 2-G and 2-H of Example 2-4 have a ceramide and glucosylceramide production-promoting effect.
  • Example 2-5 As shown in Table 2, it was confirmed that the samples 2-G and 2-H of Example 2-4 have a ceramide and glucosylceramide production-promoting effect.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the anomalous Pichia was used to replace mala (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0349). In the same manner as in Example 2-1, except that the yeast Pichia anomala) (No. 360-20-3) was used.
  • the mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight.
  • 2-J and 2-K As a sample of the ceramide/glucosylceramide production promoter of the present invention, 2-J and 2-K, the samples 2-J and 2-K were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. Stored in a refrigerator at 4 °C as an additive for cell culture.
  • the ceramide/glucose ceramide production promoting effect of the samples 2-1, 2-J, and 2-K was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia membranifaciens ( deposited in the Industrial Microbial Resources and Information Center of Cangnam University, number CICIM Y0360) was used instead of the abnormality. The same was carried out in the same manner as in Example 2-1 except that the yeast (i3 ⁇ 4/ ⁇ "oma/) (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucose ceramide production promoter sample 2-L of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Samples 2_M and 2-N which are ceramide/glucosylceramide production promoters of the present invention, were prepared by dissolving the samples 2-M and 2-N in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was used as an additive for cell culture in a refrigerator at 4 °C.
  • Example 2-7 The ceramide/glucose ceramide production promoting effect of the samples 2-L, 2-M, and 2-N was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together. As shown in Table 2, it was confirmed that the samples 2-L, 2-M, and 2-N of Example 2-6 had a ceramide production promoting effect.
  • Example 2-7 It was confirmed that the samples 2-L, 2-M, and 2-N of Example 2-6 had a ceramide production promoting effect.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the Pichia pastoris iPichia cmomala (preserved at the Industrial Microbial Resources and Information Center of Jiangnan University, CICIM Y0420) was substituted for P. angustifolia. In the same manner as in Example 2-1, except that Pichia anomala) (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the sample 2-0 of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Samples 2-P and 2-Q which are ceramide/glucosylceramide production promoters of the present invention, were prepared by dissolving the samples 2-P and 2-Q in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. , stored in a refrigerator at 4 ° C as an additive for cell culture
  • Pichia anomala (CICIM, No. CICIM Y0421, deposited in the Industrial Microbial Resources and Information Center of Jiangnan University) was replaced by Pichia anomala In the same manner as in Example 2-1, except that ⁇ lochia anomala) (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and 1 ml of the mixture was taken as the ceramide of the present invention/ Sample 2-R of the glucosylceramide production promoter was stored in a refrigerator at 4 ° C for use as an additive for cell culture.
  • the mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight.
  • 2-S, 2-T, the samples 2-S and 2-T were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. Stored in a refrigerator at 4 °C as an additive for cell culture.
  • Example 2-9 As shown in Table 2, it was confirmed that the samples 2-R, 2-S, and 2-T of Example 2-8 had a ceramide-promoting effect.
  • Example 2-9 As shown in Table 2, it was confirmed that the samples 2-R, 2-S, and 2-T of Example 2-8 had a ceramide-promoting effect.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia burtonii (CICIM, No. CICIM Y0424, deposited at the Industrial Microbial Resources and Information Center of Jiangnan University) was replaced by Abnormal Pichia. The yeast (No. 360-20-3) was cultured in the same manner as in Example 2-1.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the sample 2-U of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • the mixture of the above diluted ethanol was sonicated for 10 minutes, centrifuged at OOOO rpm for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight.
  • the sample 2-V was dissolved in 50% ethanol to prepare a 1% concentration solution, and stored in a refrigerator at 4 ° C for cell culture. Additives.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondii (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0440) was replaced. The culture was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucose ceramide production promoter sample 2-W of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • the ceramide/glucose amide production promoting effect of the sample 2-W was evaluated in the same manner as in Example 2-1, and the results are shown in Table 2 together.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondi (contained at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0326) was used instead of the abnormality. The same was carried out in the same manner as in Example 2-1 except that the yeast (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 2-X of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • the supernatant (about 70 ml) was collected, the supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucose ceramide production promoter of the present invention.
  • the sample 2- ⁇ was dissolved in 50% ethanol to prepare a 0.1% strength solution, and stored in a refrigerator at 4 ° C as an additive for cell culture.
  • Example 2 - 12 The ceramide/glucose ceramide production promoting effect of the sample 2-X, 2-Y was evaluated in the same manner as in Example 2-1, and the results are shown together in Table 2. As shown in Table 2, it was confirmed that the samples 2-X and 2-Y of Example 2-11 have a glucosylceramide production-promoting effect.
  • Example 2 - 12 The ceramide/glucose ceramide production promoting effect of the sample 2-X, 2-Y was evaluated in the same manner as in Example 2-1, and the results are shown together in Table 2. As shown in Table 2, it was confirmed that the samples 2-X and 2-Y of Example 2-11 have a glucosylceramide production-promoting effect.
  • Example 2 - 12 Example 2 - 12
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, the Pichia guilliermondii) (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0329) was used instead of the abnormality. The same was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 2-Z of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged (3000 rpm) for 10 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant temperature.
  • the sample 2-a was dissolved in 50% ethanol to prepare a 1% concentration solution, and stored in a refrigerator at 4 ° C as a cell culture. Use additives.
  • Example 2-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 2-1, Pichia guilliermondii (preserved by Jiangnan University Industrial Microbial Resources and Information Center CICIM, No. CICIM Y0414) was replaced. The culture was carried out in the same manner as in Example 2-1 except that Pichia pastoris (No. 360-20-3) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes).
  • the remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper.
  • ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent.
  • Example 3-1 Manufacture of ceramide and/or glucosylceramide production promoter
  • Candida zeylanoides purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 472) -20-1).
  • the above-mentioned strain obtained by culturing in YPD (Yeast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into a conical flask (capacity 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours.
  • the medium contained 10 w/v% okara and lw/v% glucose.
  • the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. After dilution with 2.5 times of absolute ethanol, 1 ml was taken as a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 3- ⁇ -1).
  • the crude product was treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifuged at 3000 rpm for 30 minutes, and the supernatant was recovered.
  • the supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract is dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract is adjusted to be 1 w/v% (inventive product 3-A-2), and then stored at 4 ° C for use in the subsequent Ceramide/glucose ceramide production promotion test.
  • Example 3-2
  • Candida zeylanoides was purchased from Jiangnan University and preserved at CICIM, China University Industrial Microbial Resources and Information Center, original number 406-20 -1 ).
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • the ceramide/glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-B-2) 0.1 w/v% (inventive product 3-B-3). Stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion assays.
  • Example 3-3
  • Candida parapsilosis was used (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 5 14- 20-3).
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention 3-C-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • the ceramide/glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • soybean residue fermentation extract was dissolved in 5 (%% ethanol), and the concentration thereof was adjusted to 1 w/v% (the present invention 3-C-2), and then stored at 4 ° C for the subsequent ceramide/glucose. Ceramide production promotion test. Examples 3-4
  • Ccmdi parapsilosis (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 525- 20-2).
  • the soybean residue fermentation extract was obtained in the same manner as in Example 3-1.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to 1 w/v% (3-D1 of the present invention) and 0.1 w/v% (3-D-2 of the present invention). Stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion assays. Examples 3-5
  • Candida metapsilosis is used (purchased from Jiangnan University and preserved at CICIM, China University Industrial Microbial Resources and Information Center, original number 510-19 -3 ).
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention (the present invention 3-E-1) was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • the ceramide/glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-E-2) and 0.1 w/v% (inventive product 3-E-3). Thereafter, it was stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion test.
  • Example 3-6
  • Candida parapsilosis purchased from Jiangnan University and deposited in CICIM, China University Industrial Microbial Resources and Information Center, under the accession number CICIM Y0314
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention (3-F-1 of the present invention) was obtained in the same manner as in Example 3-1, and the crude soybean obtained by the crude product was fermented and extracted.
  • the ceramide/glucose ceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean obtained by the crude product was fermented and extracted.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (the present invention 3-F-2) and 0.1 w/v% (the present invention 3-F-3). Thereafter, it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test.
  • Example 3-7
  • Example 3-1 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Candida parapsilosis ( purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0322 ). In the same manner as in Example 3-1, a crude product of the ceramide and/or glucose ceramide production promoter of the present invention (the present invention 3-G-1) was obtained, and the crude product was further purified by the crude product. As a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-G-2), 0.1 w/v% (inventive product 3-G-3). Thereafter, it was stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion test.
  • Candida boidini candida boidini (purchased from Jiangnan University and preserved in China University Industrial Microbial Resources and Information Center CICIM, Accession No. CICIM Y0366
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1 (Inventive product 3-H-1) Further, the soybean residue fermentation extract obtained by purifying the crude product is used as the ceramide/glucosylceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to 1 w/v% (the present invention, 3-H-2), and then stored at 4 Torr for use in the subsequent ceramide/glucosylceramide. A boost test was generated. Examples 3-9
  • Candida metapsilosis purchased from Jiangnan University and preserved in CICIM, China University Industrial Microbial Resources and Information Center, with the accession number CICIM Y0439
  • the soybean residue fermentation extract was obtained in the same manner as in Example 3-1.
  • soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-1-1) and 0.1 w/v% (inventive product 3-1-2). Stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion assays.
  • Example 3-10
  • Candida parapsilosis purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number C1CIM Y0443
  • the soybean residue fermentation extract was obtained in the same manner as in Example 3-1.
  • Candida utilis Ccmdidci magnoli ⁇ (purchased from Jiangnan University and preserved in
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3- ⁇ -1), 0.1 w/v% (inventive product 3-K-2). Thereafter, it was stored at 4 ° C for subsequent ceramide/glucose ceramide production promotion test.
  • Example 3-12
  • Candida maltoscO Candida maltoscO (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0207) o
  • the soybean residue fermentation extract was obtained in the same manner as in Example 3-1.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration was adjusted to 0.1 w/v% (3-L-1 of the present invention), and then stored at 4 ° C for the subsequent ceramide/ Glucose ceramide production promotion test.
  • Example 3-1 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida lipolytica (Owi3 ⁇ 43 ⁇ 4 W ⁇ to/_y/c) (purchased from Jiangnan University and deposited in the Industrial Microbial Resources and Information Center of China University of China) CICIM, deposit number CICIM Y0221).
  • a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (3- ⁇ -1 of the present invention) was obtained, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • a ceramide/glucose ceramide production promoter of the present invention it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test.
  • Example 3-14 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Candida lipolytica (Owi3 ⁇
  • Candida butyricum (Ccmdkia imtyrO (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0233)
  • the soybean residue fermentation extract was obtained in the same manner as in Example 3-1. Further, the soybean residue fermentation extract was dissolved in 5 (%% ethanol), and the concentration was adjusted to 1 w/v% (the present invention 3-N-1), and then stored at 4 ° C for the subsequent ceramide/ Glucosylceramide production promotion test. Examples 3-15
  • Candida rhagi O Candida rhagi O (purchased from Jiangnan University and deposited at the China University Industrial Microbial Resources and Information Center CICIM, accession number CICIMY0248) was used.
  • a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 3-0-1) was obtained, and the crude soybean product fermentation extract was further purified.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 3-0-2) and 0.1 w/v% (inventive product 3-0-3). Thereafter, it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test.
  • Example 3-16
  • Candidazeylanoide purchased from Jiangnan University and preserved at the China University Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0428
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • the ceramide/glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • soybean residue fermentation extract was dissolved in 50 ⁇ / ⁇ % ethanol, adjusted to a concentration of lw / v% (3-P-2 of the present invention), and stored at 4 ° C for subsequent ceramide / glucose Ceramide production promotion test.
  • Candida parapsilosis purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM) Y0325.
  • the crude product of the ceramide and/or glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • the ceramide/glucosylceramide production promoter of the present invention was obtained in the same manner as in Example 3-1, and the crude soybean product fermentation extract was obtained by further purifying the crude product.
  • soybean residue fermentation extract was dissolved in 5 (%% ethanol), and the concentration was adjusted to be lw/v%.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at a concentration of 5% at 37 ° C and C0 2 .
  • the cell culture was carried out under the conditions until the cell converence was 80 to 90%.
  • Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%.
  • the cells were passaged to a 12-well plate (2 ml per well) at IX 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • the cells were washed twice with 2 ml PBS (phosphate buffer), and 1 ml of PBS was added to the wells, and the cells were collected into a test tube by a cell harvester (cell shovel) for subsequent ceramide/glucose ceramide and protein analysis.
  • PBS phosphate buffer
  • cell harvester cell shovel
  • the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro.
  • the relative values of Cer/Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 3.
  • Cer/Pro Ceramic amide amount (g/ml) / protein content g/ml)
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content ⁇ g/ml) table 3
  • Example 3-1 As shown in Table 3, the inventive product obtained in Example 3-1, Examples 3-6 to 3-7, and Example 3-17 had both a ceramide production promoting effect and a glucosylceramide.
  • the glucosylceramide production-promoting effect of the present invention obtained in Example 3-2, Examples 3-9 to 3-13, and Example 3-15 was remarkable, and Example 3 _3 to 3- 5.
  • the ceramide production promoting effect of the present invention obtained in Examples 3-8, Examples 3-14 and Examples 3-16 was remarkable.
  • the ceramide/glucose ceramide production promoting agent of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.
  • the ceramide/glucosylceramide production promoter obtained in the above Examples 3-1 to 3-17 was directly analyzed by TLC without cell culture, and as a result, no ceramide and glucosylceramide were present in the present invention.
  • Debaryomyces hansenii was used (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 476 -20-1).
  • the above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours.
  • the medium contained 10 w/v% okara and 1 w/ v % glucose.
  • the bean dregs are prepared by soaking the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the cells are recovered after centrifugation.
  • the supernatant was added to the culture supernatant after the recovery, and 2.5 times of absolute ethanol was added thereto, and the mixture was pulverized by an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifuged at 3000 rpm for 30 minutes, and the supernatant was recovered.
  • the supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to be lw/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.
  • Example 4-2
  • Example 4-1 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, Debatyomyces hansenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, original number 5) 14-20-4).
  • the soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.
  • Example 4-3
  • Example 4-4 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Debaryomyces hansenii (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM) Y0286 A soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 C for subsequent ceramide/glucose ceramide production promotion test. 4-5
  • soybean residue fermentation extract was dissolved in 5% ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.
  • Example 4-7 As a microorganism that produces ceramide/glucose ceramide production promoter from bean dregs, use Debaryomyces hansenii) (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0290 ).
  • the soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to be lw/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.
  • Example 4-8
  • Example 4 - 1 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, use B. vannamei ebaryomyces vanrijiae (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0198 adopted and implemented Example 4 - 1 The same method was used to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration of the soybean residue fermentation extract was adjusted to 0.1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.
  • Example 4-9
  • Example 4-1 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Debaiyomyces hansenii (purchased from Jiangnan University and deposited in CICIM, China University Industrial Microbial Resources and Information Center, CICIM) Y035 1 A soybean residue fermentation extract was obtained in the same manner as in Example 4-1 as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5% ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 1 w/v%, and then stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test. (ceramide/glucose ceramide production promotion test)
  • ⁇ Keratinocytes culture> activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium), and cells were cultured at 37 ° C and a CO 2 concentration of 5%. The culture is maintained until the cell aggregation density (concentration) is 80 to 90%. Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at 1 ⁇ 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro.
  • the relative values of Cer Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 4.
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content (g/ml)
  • Example 4-8 0.99 0.91 1.13 1.04
  • Example 4-9 1.53 1.40 1.35 1.23
  • the inventive product obtained in Example 4-1 and Example 4-9 has both a ceramide production promoting effect and a glucosylceramide production promoting effect;
  • the ceramide production-promoting effect of the present invention obtained in Example 4-2 and Examples 4-4 to 4-7 was remarkable, and the glucose of the present invention obtained in Example 4-3 and Example 4-8 was obtained.
  • the ceramide production promoting effect is remarkable. Therefore, it was confirmed that the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect. (Test for the absence of ceramide and glucosylceramide in the ceramide/glucosylceramide production promoter of the present invention)
  • the ceramide/glucosylceramide production promoter obtained in the above Examples 4- 1 to 4-9 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide and glucosylceramide present in the present invention.
  • the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. .
  • the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. It is crushed, filtered and the liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentration of the okara and glucose in the mixture was respectively 10 w/ V . / ⁇ Q lw/v% , the mixture was used as a medium. '
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times absolute ethanol, and 1 ml of the mixture was taken as the ceramide of the present invention.
  • Sample 5-A of the glucosylceramide production promoter was stored in a 4 C refrigerator for use as an additive for cell culture.
  • the diluted ethanol solution was sonicated for 10 minutes, centrifuged at OOOO rpm for 30 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight.
  • the dried product was used as the sample 5-B, 5-C of the ceramide and/or glucosylceramide production promoter of the present invention, and the samples 5-B and 5-C were each dissolved in 50% ethanol to prepare 1%.
  • a 0.1% strength solution was stored in a refrigerator at 4 ° C as an additive for cell culture.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at 37 ° C and a CO 2 concentration of 5%. The cells were cultured to a concentration of 80 to 90%. After passage, the cells were passaged into a 175 cm 2 square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per well, and the cell concentration was IX 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • methanol: chloroform 2.5 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant a and a precipitate b.
  • the supernatant a was used for the analysis of the amount of ceramide/glucose amide, and the precipitate b was used for the analysis of the protein content.
  • the ceramide (or glucosylceramide) standard and sample solution are spotted onto a dry TLC plate and chromatographed with CHC13 : CH30H: CH3COOH (190: 9: 1 v/v/v) as a layering agent.
  • the layer is applied to the top of the sheet and the spreader is blown dry with a blower; the above steps are repeated once.
  • CHCB: CH30H: C3H60 76: 20: 4 v/v/v
  • Example 5-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 5-1, the Cryptococcus hungariciis (CICIM deposited in the Industrial Microbial Resources and Information Center of Jiangnan University, number CICIM Y0203) was used instead of the Loren Hidden Ball. The culture was carried out in the same manner as in Example 5-1 except for the yeast Cryptococc laurentii) (No. CICIM Y0232).
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as a sample 5-D of the ceramide/glucose ceramide production promoter of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Example 5-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 5-1, the C. cerevisiae i Cryptococcus laurentii (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0218) was used instead of The culture was carried out in the same manner as in Example 5-1 except that Cryptococcus laurentii (No. CICIM Y0232) was used. The resulting culture was then centrifuged (3000 rpm, 10 minutes).
  • the remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 5-G of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Example 5-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 5-1, the Cryptococcus hungaricus (CICIM, numbered CICIM Y0219, deposited at the Jiangnan University Industrial Microbial Resources and Information Center, CICIM Y0219) was used instead of the Loren Hidden Ball.
  • the yeast Cryptococcus laurentii) No. CICIM Y0232 was cultured in the same manner as in Example 5-1.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixture was taken as the ceramide/glucose ceramide production promoter sample 5-1 of the present invention, and stored in a refrigerator at 4 ° C for use in cell culture. Additives.
  • ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent.
  • Example 6-1
  • the above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium at 30 Torr.
  • the culture was carried out for 72 hours.
  • the medium contained 10 w/v% okara and lw/v% glucose.
  • the bean dregs are prepared by immersing the soybeans in water for 12 hours to sufficiently absorb the moisture of the soybeans, and then grinding and damaging the water-absorbing sun beans, filtering and removing the liquid components, and then draining the remaining residue to obtain.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added, and the mixture was pulverized for 10 minutes by an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.), centrifuged at 3000 rpm for 30 minutes, and the supernatant was blown back.
  • an ultrasonic treatment apparatus manufactured by Branson Co., Ltd.
  • the supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide and/or glucosylceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 50% by weight of ethanol, and the concentration of the soybean residue fermentation extract was adjusted to 0.1 w/v% (the present invention 6-A-1), and then stored at 4 ° C for use in the subsequent Ceramide/glucose ceramide production promotion test.
  • Example 6-2
  • Example 6-3 1 001877 As a microorganism for the production of ceramide/glucose ceramide production promoter from bean dregs, using Hansenula polymorpha iHansenula polymorph ⁇ (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0257).
  • the soybean residue fermentation extract was obtained in the same manner as in Example 6-1, and the ceramide and/or glucosylceramide production promoter of the present invention was further dissolved in 5 (%% ethanol) to adjust the concentration thereof. After 0.1 w/v% (inventive product 6-C-1), it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test.
  • Example 6-4 1 001877 As a microorganism for the production of ceramide/glucose ceramide production promoter from bean dregs, using Hansenula polymorpha iHansenula polymorph
  • Haemda polymorpha is used (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0122) ).
  • the soybean residue fermentation extract was obtained in the same manner as in Example 6-1 as a ceramide and/or glucosylceramide production promoter (Inventive product 6-D-1) of the present invention, and further, 50 ⁇ / ⁇ % ethanol was used. Dissolving the soybean residue fermentation extract and adjusting the concentration to lw/v%
  • Example 6-6 As a microorganism that produces a ceramide/glucose ceramide production promoter from bean dregs, Hansenula polymorpha) (purchased from Jiangnan University and preserved in China Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0135) .
  • the soybean residue fermentation extract was obtained in the same manner as in Example 6-1, and was stored as a ceramide and/or glucosylceramide production promoter (Inventive product 6-E-1) of the present invention, and stored at 4 ° C, For subsequent ceramide/glucose ceramide production promotion experiments.
  • Example 6-6
  • Example 6 As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Hansenula sylvestris iHansenula ciferrii (purchased from Jiangnan University and deposited at the China University Industrial Microbial Resources and Information Center CICIM, accession number CICIM Y0150) was used.
  • the soybean residue fermentation extract was obtained in the same manner as in Example 6-1 as a ceramide and/or glucosylceramide production promoter of the present invention, and further, the soybean residue fermentation extract was dissolved in 50 v/v% ethanol to adjust the concentration thereof. After lw/v% (product 6-F-1 of the present invention), it was stored at 4 ° C for the subsequent ceramide/glucose ceramide production promoting test.
  • Example 6-7 As a microorganism for producing a ceramide/glucose ceramide production promoter from bean dregs, Hansenula sy
  • activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium), and cells were cultured at 37 ° C and a CO 2 concentration of 5%. The culture is maintained until the cell aggregation density (confluence) is 80 to 90%. Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at 1 ⁇ 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content (g/ml) Table 6
  • Example 6-1, Example 6-3, and Example 6 The ceramide production-promoting effect of the present invention obtained in 6-6 was remarkable, and the glucosylceramide production-promoting effect of the present invention obtained in Example 6-4 and Example 6-7 was remarkable. Therefore, it was confirmed that the ceramide and/or glucosylceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect. (Test for the absence of ceramide and glucosylceramide in the ceramide and/or glucosylceramide production promoter of the present invention)
  • the ceramide and/or glucosylceramide production promoter obtained in the above Examples 6-1 to 6-7 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide and glucosylceramide in the present invention. presence.
  • Bombyx serrata (Bulle tsugcw (purchased from Jiangnan University and preserved at CICIM, China University Industrial Microbial Resources and Information Center, preserved) No. CICIM Y0259 ) o
  • the above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours.
  • the medium contained 10 w/v% okara and l w/v% glucose.
  • the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. After diluting with 2.5 times of absolute ethanol, 1 ml was taken as a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 7-A).
  • the crude product was treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifuged at 3000 rpm for 30 minutes, and the supernatant was recovered. Filter about 70 ml of the supernatant with a filter paper, and then dry the filtrate to a constant weight in a vacuum dryer below 40 ° C to obtain beans.
  • the slag fermentation extract is used as a ceramide and/or glucosylceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 50 Wv% ethanol, and the concentration thereof was adjusted to 1 w/v% (inventive product 7-B), 0.1 w/v% (inventive product 7-C), and stored at 4°. C, for subsequent ceramide/glucose ceramide production promotion test.
  • activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium), and cells were cultured at 37 ° C and a CO 2 concentration of 5%. The culture is maintained until the cell aggregation density (concentration) is 80 to 90%. Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at 1 ⁇ 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro.
  • the relative values of Cer/Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to be 100 (respectively referred to as Cer/Well and GlyCer/Well, respectively, relative to the relative amount of the control). Also shown in Table 7.
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content (g/ml) Table 7
  • the present invention 7-C 4.23 3.95 0.96 0.90 7-1
  • the ceramide production-promoting effect of the present invention 7-A to 7-C obtained in Example 7-1 was remarkable. Therefore, it has been confirmed that the ceramide and/or glucose amide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.
  • the amide and/or glucosylceramide production promoter obtained in the above Example 7-1 was directly analyzed by TLC without cell culture, and the results showed that ceramide and glucosylceramide were not present in the present invention.
  • the culture medium was prepared as follows: Soy soybeans were immersed in water for 12 hours to allow the soybeans to sufficiently absorb moisture, and then the soybeans which absorbed the moisture were ground. It is crushed, filtered and the liquid component is removed, and the remaining residue is drained to obtain okara. The okara was mixed with glucose and water, and the concentrations of the okara and glucose in the mixture were respectively 10% and 1 w/v%, and the mixture was used as a medium.
  • Rhodotonda mucilaginosa preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0250, and cultured at 30 °C. day.
  • the diluted ethanol solution was sonicated for 10 minutes, centrifuged at 80 rpm for 30 minutes, and the supernatant (about 70 ml) was collected. The supernatant was filtered through a filter paper to obtain a filtrate, and the filtrate was dried in a vacuum dryer to a constant weight.
  • the dried product was used as the sample of the ceramide and/or glucosylceramide production promoter of the present invention 8-B, 8-C, and the samples 8-B and 8-C were each dissolved in 50% ethanol to prepare 1%.
  • a 0.1% strength solution was stored in a refrigerator at 4 ° C as an additive for cell culture.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at 37 ° C and a CO 2 concentration of 5%. The cells were cultured to a concentration of 80 to 90%. After passage, the cells were passaged into a 175 cm 2 square vial and the cells were cultured to a concentration of 80 to 90%. The passaged human epidermal cells were then inoculated into a 12-well plate at a volume of 2 ml per cell at a cell concentration of 1 ⁇ 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • methanol: chloroform 2.5 ml: 1.25 ml of a solvent was added, and the mixture was shaken for 20 minutes, and centrifuged (3000 rpm, 5 minutes) to obtain a supernatant a and a precipitate b.
  • the supernatant a was used for the analysis of the amount of ceramide/glucose amide, and the precipitate b was used for the analysis of the protein content.
  • CHCB CH30H: CH3COOH ( 190: 9: 1 v/v/v) as a layering agent.
  • the layer is applied to the top of the sheet and the spreader is blown dry with a blower; the above steps are repeated once.
  • CHCB: CH30H: C3H60 76: 20: 4 v/v/v
  • Example 8.1 In the preparation of the ceramide/glucose ceramide production promoter of Example 8.1, the sticky red circle was replaced by Rhodotorula mucilaginosa ( deposited in the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0433). The yeast (Rhodotorula mucilaginosa ⁇ ) (No. CICIM Y0250) was cultured in the same manner as in Example 8.1.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 8-D of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Samples 8-E and 8-F which are ceramide/glucosylceramide production promoters of the present invention, and samples 8-E and 8-F were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture.
  • Example 8.1 In the preparation of the ceramide/glucose ceramide production promoter of Example 8.1, the viscosity was replaced by Rhodotorula mucilaginosa (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0430). The culture was carried out in the same manner as in Example 8-1 except that Rhodotonda mucilaginosa) (No. CICIM Y0250) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). Remaining supernatant
  • the solution was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was used as the ceramide/glucose ceramide production promoter sample 8-G of the present invention, and stored in a refrigerator at 4 ° C for use as an additive for cell culture.
  • Example 8-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, R. erythromycin Rhodoto la glutinis (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0148) was used instead of the sticky red yeast. (Rhodotorula mucilaginosa) (No. CICIM Y0250) was cultured in the same manner as in Example 8.1.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes).
  • the remaining supernatant was diluted with 2.5 times absolute ethanol, and the diluted mixture of the above anhydrous ethanol was sonicated for 10 minutes, centrifuged (3000 rpm) for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered.
  • the filtrate was filtered, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucosylceramide production promoter of the present invention.
  • 8-H and the sample 8-H was dissolved in 50% ethanol to prepare a concentration of 1%.
  • the solution was stored in a refrigerator at 4 ° C as an additive for cell culture.
  • Example 8-5 As shown in Table 8, it was confirmed that the sample 8-H of Example 8-4 had a glucosylceramide production promoting effect.
  • Example 8-5 As shown in Table 8, it was confirmed that the sample 8-H of Example 8-4 had a glucosylceramide production promoting effect.
  • Example 8-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, the viscous red circle was replaced with the viscous red yeast iRhodotonda mucilaginosa ⁇ (preserved at the Industrial Microbial Resources and Information Center of Cangnam University, CICIM Y0324). Yeast Rhodotorula mucilaginos O (No. CICIM Y0250), in addition to Example 8-1 was also cultured in the same manner.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes).
  • the remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper.
  • the filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer to obtain samples 8-1 and 8-J of the ceramide/glucosylceramide production promoter of the present invention, and samples 8-1 and 8-J were respectively 50%.
  • the solution was dissolved in ethanol to prepare a solution having a concentration of 1% and 0.1%, and stored in a refrigerator at 4 °C as an additive for cell culture.
  • Rhodotoru mucilaginosa preserved at Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0337
  • the culture was carried out in the same manner as in Example 8-1 except that Rhodotoruh mucilaginosa (No. CICIM Y0250) was used.
  • the resulting culture was then centrifuged to GOOO rpm for 10 minutes).
  • the remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 8-K of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Rhodotoruh mucilaginosa (reserved in Jiangnan University)
  • the culture microbial resources and information center CICIM (No. CICIM Y0394) was cultured in the same manner as in Example 8-1 except that Rhodotorula muci ginosa (No. CICIM Y0250) was used.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes).
  • the remaining supernatant was diluted with 2.5 times of absolute ethanol, and the mixture diluted with the above anhydrous ethanol was sonicated for 10 minutes, centrifuged at 80 rpm for 10 minutes, and the supernatant (about 70 ml) was collected, and the supernatant was filtered through a filter paper.
  • the filtrate was obtained, and the filtrate was dried to a constant weight in a vacuum dryer to obtain a sample of the ceramide/glucosylceramide production promoter of the present invention.
  • 8-L and the sample 8-L was dissolved in 50% ethanol to prepare a 1% concentration.
  • the solution was stored in a refrigerator at 4 °C as an additive for cell culture.
  • Example 8-1 In the preparation of the ceramide/glucose ceramide production promoter of Example 8-1, R. sylvestris Rhodotoru mucilaginosa (preserved at Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIMY0418) was used instead of the viscous red yeast (Rhodotonda mucilaginosa (No. CICIM Y0250) was cultured in the same manner as in Example 8-1.
  • the resulting culture was then centrifuged (3000 rpm, 10 minutes). The remaining supernatant was diluted with 2.5 times of absolute ethanol, and 1 ml of the mixed solution was taken as the ceramide/glucose ceramide production promoter sample 8-M of the present invention, and stored in a refrigerator at 4 ° C for use as a cell culture addition. Things.
  • Samples 8-N and 8-0 which are ceramide/glucosylceramide production promoters of the present invention, and samples 8-N and 8-0 were each dissolved in 50% ethanol to prepare a solution having a concentration of 1% and 0.1%. It was stored in a refrigerator at 4 °C as an additive for cell culture.
  • the ceramide/glucose ceramide production promoting effect of the samples 8-M, 8-N, and 8-0 was evaluated in the same manner as in Example 8.1, and the results are shown in Table 8.
  • Example 8-9 As shown in Table 8, it was confirmed that the samples 8-M, 8-N, and 8-0 of Example 8-8 have a ceramide and glucosylceramide production-promoting effect.
  • Example 8-9 As shown in Table 8, it was confirmed that the samples 8-M, 8-N, and 8-0 of Example 8-8 have a ceramide and glucosylceramide production-promoting effect.
  • Rhodotorula mucilaginosa preserved at Jiangnan University Industrial Microbial Resources and Information Center CICIM, number CICIM Y0423 was used instead of viscous red.
  • the culture was carried out in the same manner as in Example 8-1 except that Rhodotorula mucilaginoscO (No. CICIM Y0250) was used.
  • Rhodotorula The present invention 0.04 0.04 0.94 1.02 mucilaginosa ) 8-E
  • the invention is 0.05 0.07 0.85 1.1 1 8-F
  • the invention is 2.41 1.94 2.52 2.03 8-0
  • ceramide or glucosylceramide is not present in the present invention itself, but the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoting effect. Use of the agent.
  • Example 9-1
  • the above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours.
  • the medium contained 10 w/v% okara and lw/v% glucose.
  • the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added to obtain a crude product of the ceramide and/or glucosylceramide production promoter of the present invention (inventive product 9-A), which was stored at 4 ° C, and was used for the subsequent ceramide/glucose nerve. Amide production promotion test.
  • Example 9-2 a crude product of the ceramide and/or glucosylceramide production promoter of the present invention
  • the supernatant was filtered by a filter paper to a volume of about 70 ml, and the filtrate was dried in a vacuum dryer at 40 ° C or lower to a constant weight to obtain a bean slag as a ceramide/glucose ceramide production promoter of the present invention. Fermented extract.
  • soybean residue fermentation extract was dissolved in 5 (%% ethanol), adjusted to a concentration of 0.1 w/v% (inventive product 9-B), and stored at 4 ° C for subsequent ceramide/glucose nerves. Amide production promotion test.
  • Example 9-3
  • the present invention product 9-C was obtained in the same manner as in Example 9-1, and then treated with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.). The crude product was centrifuged for 10 minutes at 3000 rpm for 10 minutes, and the supernatant was recovered.
  • the supernatant was filtered through a filter paper to a volume of about 70 ml, and the filtrate was dried to a constant weight in a vacuum dryer at 40 ° C or lower to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% ethanol), adjusted to a concentration of 1 w/v% (inventive product 9-D), and stored at 4 ° C for the subsequent ceramide/glucosylceramide. Produce a boost test.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at a concentration of 5% C0 2 at 37 ° C.
  • the cell culture was carried out under the conditions until the cell converence was 80 to 90%.
  • Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%.
  • the cells were passaged to a 12-well plate (2 ml per well) at IX 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • only 5 ( ⁇ % ethanol (without adding ceramide/glucose ceramide production promoter) was added as a control, and cultured for 72 hours under the same culture conditions. After 72 hours of cultivation, the supernatant culture solution was discarded.
  • Cer/Pro amount of amide (g/ml) / protein content (g/ml)
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content (g/ml) Table 9
  • Example 9-1 and Example 9-3 As shown in Table 9, the glucosylceramide production-promoting effect of the present invention obtained in Example 9-1 and Example 9-3 was remarkable, and the ceramide production of the present invention obtained in Example 9-2 was promoted. The effect is remarkable.
  • the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect. .
  • the ceramide/glucosylceramide production promoter obtained in the above Examples 9-1 to 9-3 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide or glucosylceramide present in the present invention.
  • the above-mentioned strain obtained by culturing in YPD (Yast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium, at 30 Incubate at °C for 72 hours.
  • the medium contained 10 w/v% okara and lw/v% glucose.
  • the bean dregs are prepared by immersing the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added, followed by treatment with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifugation at 3000 rpm for 30 minutes, and the supernatant was recovered.
  • an ultrasonic treatment apparatus manufactured by Branson Co., Ltd.
  • the supernatant was filtered with a filter paper to a volume of about 70 ml, and the filtrate was dried in a vacuum dryer at 40 ° C or lower to constant weight to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 5 (% ethanol), adjusted to a concentration of 1 w/v% (inventive product 10-A), and stored at 4 ° C for the subsequent ceramide/glucose nerve. Amide production promotion test.
  • Example 10-2
  • soybean residue fermentation extract was dissolved in 50% by weight of ethanol, adjusted to a concentration of 0.1 w/v% (inventive product 10-B), and stored at 4 ° C for subsequent ceramide/glucosylceramide Produce a boost test.
  • Example 10-3
  • soybean residue fermentation extract was dissolved in 5 (% by weight of ethanol), and the concentration thereof was adjusted to be 1 w/v% (inventive product 10_C) and 0.1 w/v% (inventive product 10-D), and then stored in 4 At °C, it was used in subsequent ceramide/glucose ceramide production promotion tests.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at 37 °C.
  • the cell culture was carried out under the condition that the concentration of C0 2 was 5% until the cell aggregation density (confluence) was 80 to 90%.
  • Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%. Then, the cells were passaged to a 12-well plate (2 ml per well) at IX 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • the cells were washed twice with 2 ml PBS (phosphate buffer), and 1 ml of PBS was added to the wells, and the cells were collected into a test tube by a cell harvester (cell shovel) for subsequent ceramide/glucose ceramide and protein analysis.
  • PBS phosphate buffer
  • cell harvester cell shovel
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content g/ml) Table 10
  • Example 10-1 As shown in Table 10, the present invention obtained in Example 10-1 has both a ceramide production promoting effect and a glucosylceramide production promoting effect, and the examples obtained in Examples 10-2 and 10-3.
  • the glucosylceramide production promoting effect of the invention product is remarkable.
  • the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.
  • the ceramide/glucose ceramide production promoter obtained in the above Examples 10-1 to 10-3 was directly analyzed by TLC without cell culture, and the results showed that there was no ceramide and glucosylceramide present in the present invention.
  • rr OW a lipolytica ( purchased from Jiangnan University and deposited in CICIM, China University Industrial Microbial Resources and Information Center, is used. Deposit number CICIM Y0311)o
  • YPD Yeast Extract Peptone Dextrose Medium
  • the above-mentioned strain obtained by culturing in YPD (Yeast Extract Peptone Dextrose Medium) medium at 30 ° C was inoculated into an Erlenmeyer flask (capacity: 300 ml) containing 100 ml of a ceramide/glucosylceramide production promoter production medium at 30 Torr. The culture was carried out for 72 hours.
  • the medium contains 10 w/v% okara and ⁇ % glucose.
  • the bean dregs are prepared by soaking the soybeans in water for 12 hours, so that the soybeans sufficiently absorb the water, and then the soybeans which have absorbed the water are ground and crushed, filtered, and the liquid components are removed, and the remaining residue is drained and obtained.
  • the cells are removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant after centrifugation is recovered, and the culture supernatant after the recovery is collected. 2.5 times of absolute ethanol was added, followed by treatment with an ultrasonic treatment apparatus (manufactured by Branson Co., Ltd.) for 10 minutes, centrifugation at 3000 rpm for 30 minutes, and the supernatant was recovered.
  • an ultrasonic treatment apparatus manufactured by Branson Co., Ltd.
  • the supernatant was filtered through a filter paper to a thickness of about 70 m, and the filtrate was dried in a vacuum dryer at 40 ° C or lower to a constant weight to obtain a soybean residue fermentation extract as a ceramide/glucose ceramide production promoter of the present invention.
  • soybean residue fermentation extract was dissolved in 50 v/v% ethanol, adjusted to a concentration of 1 w/v%, and stored at 4 ° C for the subsequent ceramide/glucose ceramide production promotion test.
  • normal human activated keratinocytes (Cascade Co., Ltd.) were used as primary cells in a 75 cm 2 flask (2 ml of growth factor-containing medium) at a concentration of 5% C0 2 at 37 ° C.
  • the cell culture was carried out under the conditions until the cell aggregation density (concentration) was 80 to 90 ° /. .
  • Cells were passaged to 175cm 2 flasks side cultured cells to a concentration of 80 ⁇ 90%. Then, it was passaged to a 12-well plate (2 ml per well) at 1 ⁇ 10 5 cells/ml, and the cells were further cultured to a concentration of 80 to 90%.
  • the obtained values are respectively divided by the respective protein contents to obtain Cer/Pro and GlyCer/Pro.
  • the relative values of Cer/Pro and GlyCer/Pro of the present invention were determined by using Cer/Pro and GlyCer/Pro of the control as 1. Further, the amount of ceramide and glucosylceramide in each well was determined by taking the amount of protein in each well of the control to 100 (respectively referred to as Cer/Well and GlyCer/Well, relative amounts relative to the control). Also shown in Table 11.
  • GlyCer/Pro Glucose ceramide amount (g/ml) / protein content ( ⁇ ⁇ / ⁇ 1 ) Table 1 1 As shown in Table 11, the glucosylceramide production-promoting effect of the present invention obtained in Example 11-1 was remarkable.
  • the ceramide/glucose ceramide production promoter of the present invention has a ceramide production promoting effect and/or a glucosylceramide production promoting effect.
  • the amide/glucosylceramide production promoter obtained in the above Example 11-1 was directly analyzed by the TLC method without cell culture, and the results showed that there was no ceramide and glucosylceramide present in the present invention.
  • the present invention itself has no ceramide or glucosylceramide, but has a ceramide production promoting effect and/or a glucosylceramide production promoting effect, and can be used as a ceramide and/or glucosylceramide production promoter. .

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Abstract

L'invention concerne un procédé de production d'un promoteur à partir de céramide et/ou de glucosylcéramide, qui comprend les étapes consistant à: mettre en culture des levures dans un milieu de culture contenant des résidus de haricots et des glucides; et extraire du surnageant les promoteurs produits par le céramide et/ou le glucosylcéramide. Ce procédé peut utiliser diverses matières disponibles comme milieu de culture, et permet d'obtenir de manière peu coûteuse et efficace, au moyen de levure, les promoteurs produits par le céramide et/ou le glucosylcéramide.
PCT/CN2011/001877 2010-11-09 2011-11-08 Procédé de production d'un promoteur à partir de céramide et/ou de glucosylcéramide Ceased WO2012062043A1 (fr)

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CN201110289008.0A CN102994555B (zh) 2011-09-15 2011-09-15 神经酰胺和/或葡萄糖神经酰胺产生促进剂的制造方法
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10259135A (ja) * 1997-01-17 1998-09-29 Kanebo Ltd セラミド合成促進剤
JPH11322534A (ja) * 1998-05-14 1999-11-24 Kanebo Ltd セラミド合成促進剤
CN101583345A (zh) * 2006-11-02 2009-11-18 美露香株式会社 神经酰胺合成促进剂、化妆品、皮肤外用剂、抗老化方法和皱纹改善方法
CN101653561A (zh) * 2008-08-18 2010-02-24 复旦大学附属中山医院 神经酰胺产生促进剂
CN101653537A (zh) * 2008-08-18 2010-02-24 复旦大学附属中山医院 神经酰胺产生促进剂

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10259135A (ja) * 1997-01-17 1998-09-29 Kanebo Ltd セラミド合成促進剤
JPH11322534A (ja) * 1998-05-14 1999-11-24 Kanebo Ltd セラミド合成促進剤
CN101583345A (zh) * 2006-11-02 2009-11-18 美露香株式会社 神经酰胺合成促进剂、化妆品、皮肤外用剂、抗老化方法和皱纹改善方法
CN101653561A (zh) * 2008-08-18 2010-02-24 复旦大学附属中山医院 神经酰胺产生促进剂
CN101653537A (zh) * 2008-08-18 2010-02-24 复旦大学附属中山医院 神经酰胺产生促进剂

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