JP5611186B2 - Method for producing ceramide production promoter - Google Patents

Description

  The present invention relates to a method for producing a ceramide and / or glucosylceramide production promoter capable of promoting the production of ceramide and / or glucosylceramide in the skin.

The skin has the ability to produce ceramide and glucosylceramide. In the skin, particularly in the horny stratum corneum, ceramide synthesized and secreted by epidermal cells accumulates and is the main component of intercellular lipids. Ceramide synthesized in skin epidermal cells is temporarily stored in the form of glucosylceramide or sphingomyelin, then discharged out of the cell, and the action of β-glucocerebrosidase and sphingomyelinase It becomes ceramide again and functions as an intercellular lipid. Ceramide and glucosylceramide in the skin have physiological effects such as improving the skin moisturizing function and the skin barrier function.

  However, in recent years, the skin tends to dry out due to the influence of the environment, the climate, and the like. Therefore, the ability to improve the skin moisturizing function by producing the skin's own ceramide and the like is not sufficient.

  Therefore, a method of replenishing the skin with ceramide from the outside can be considered. For example, Patent Document 1 discloses a method for producing glucosylceramide by culturing a microorganism belonging to the genus Candida. The moisture retaining function of the skin can be improved by applying the glucosylceramide to human dry skin. Since the method is a method for directly obtaining glucosylceramide that is present in a large amount in cultured cells, the ceramide skeleton of glucosylceramide synthesized by the method has two double bonds, and Since it has a branch, it is different from the ceramide skeleton of ceramides existing in the human body. Therefore, when such a glucosylceramide obtained directly from the body of a microorganism is applied to human skin, it cannot be applied to the skin, and there is a risk of causing problems regarding safety in use.

  Patent Document 6 reports a method for obtaining acetic acid ceramide from acetic acid bacteria. However, there is a problem that the ceramide obtained by this method cannot be applied to human skin as described above. Moreover, in such a method, since the process of extracting from a microbial cell and obtaining ceramide etc. is required, the process becomes complicated and the problem that cost also exists also exists.

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

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

  Therefore, recently, research on producing substances aimed at promoting the production of epidermal ceramide in the stratum corneum of the skin has been actively promoted. Here, as a substance that can promote the generation of ceramide, ceramide production promoters containing extracts such as Mannentake, Bee Pollen, and Senkyu have been reported (Patent Documents 2 to 4). However, all of these ceramide production promoters are extracted from plant raw materials, and this extraction operation takes a long time, and the availability of natural resources is limited. Therefore, the production cost is high.

  Patent Document 5 discloses a ceramide synthesis promoter comprising a fungus culture containing nicotinic acid and / or nicotinamide, which can activate ceramide synthesis of epidermal cells within the skin surface layer, It is disclosed that the fungal culture may be a lactic acid bacteria culture, a bifidobacteria culture, a mushroom cell culture, or a yeast culture. However, since the medium used for preparing the ceramide synthesis promoter substantially contains nicotinic acid and / or nicotinamide as an active ingredient, the production cost is high.

  On the other hand, it is known that bacteria of a plurality of genera such as Bacillus genus, Lactobacillus genus, Acetobacter genus, Leuconostoc genus and the like are widely used in food industries.

  Bacillus microorganisms (bacteria) are universally present in water and soil. Among them, there are many species adapted to various extreme environments such as high pH, low temperature, high salt concentration, and high pressure. The genus Bacillus includes extremely useful species such as Bacillus subtilis, which are used for production of various enzymes (for food, detergents, and other various industrial enzymes).

  In addition, many microorganisms belonging to the genus Lactobacillus are present in the digestive tract of humans and animals, and are separated from their stool and easily separated from the field. A microorganism belonging to the genus Lactobacillus has long been used for the production of yogurt and the like.

  A microorganism belonging to the genus Acetobacter can oxidize ethanol to acetic acid and further oxidize it to carbon dioxide. Microorganisms of the genus Acetobacter are generally distributed on beer, fruit liquor and flowers. Microorganisms of the genus Acetobacter can cause the deterioration of alcohol with low alcohol, and can be used to brew vinegar.

  In addition, microorganisms of the genus Leuconostoc are widely distributed in plants, dairy products and other foods. Leuconostoc mesenteroides are important bacteria in the genus Leuconostoc. Studies have confirmed that Leuconostoc mesenteroides can ferment sugars to produce a variety of acids and alcohols, and have high acid-producing ability, antioxidant ability, and antagonistic pathogens. Leuconostoc mesenteroides bacteria have already been widely applied to flavoring agents, plasma substitutes, micro-ecological preparations and the like.

  However, conventionally, it has not yet been reported that a ceramide or a glucosylceramide production promoter is efficiently produced at low cost by bacteria (particularly, bacteria of each genus described above).

JP 2010-22217 A JP 2005-194240 A JP 2010-70499 A JP 2010-150237 A JP-A-9-194383 JP 2006-345796 A

  This invention makes it a subject to provide the method of manufacturing a ceramide and / or a glucosyl ceramide production promoter inexpensively and efficiently.

  As a result of intensive studies to solve the above problems, the present inventors have been able to obtain ceramide and / or glucosylceramide production promoters inexpensively and efficiently by culturing bacteria in a medium containing soybean residue and saccharides. And have led to the present invention.

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

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

  The bacterium used in the present invention may be any one selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Acetobacter, and bacteria belonging to the genus Leuconostoc.

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

  In the production method of the present invention, bacteria are cultured in a medium containing soybean residue and saccharide, and a ceramide and / or glucosylceramide production promoter is collected from the culture supernatant.

  The bacterium used in the present invention may be any one selected from bacteria belonging to the genus Bacillus, bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Acetobacter, and bacteria belonging to the genus Leuconostoc.

The microorganisms belonging to the genus Bacillus used in the present invention include Bacillus subtilis, Bacillus acidicola, Bacillus gibsonii, Bacillus licheniformis, Bacillus alcalophilus, Bacillus cereus, Bacillus pumilus, Bacillus sphaericus, Bacillus diffusus, Bacillus globigii, Bacillus globigii, Bacillus globigii, Bacillus globigii, Examples include Bacillus megaterium, Bacillus mucilaginosus, Bacillus mycoides, Bacillus thuringiensis, Bacillus circulans, Bacillus mucilaginosus, Bacillus stearothermophilus and the like.
Among these, Bacillus subtilis, Bacillus acidicola, Bacillus gibsonii, and Bacillus licheniformis are preferable from the viewpoint that the obtained component has a higher ceramide and / or glucosylceramide production promoting effect.

The microorganisms belonging to the genus Lactobacillus used in the present invention include Lactobacillus brevis, Lactobacillus farciminis, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus coryniformi, Lactobacillus curvatus, Lactobacillus alimentarius, Lactobacillus ractnocillus, Lactobacillus ractus, Lactobacillus Examples include Lactobacillus murinus, Lactobacillus fermentum, and Lactobacillus helveticus.
Among these, Lactobacillus brevis, Lactobacillus farciminis, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus coryniformi, Lactobacillus curvatus, Lactobacillus alimentarius is preferable from the viewpoint that the obtained component has a higher ceramide and / or glucosylceramide production promoting effect.

Examples of microorganisms belonging to the genus Acetobacter used in the present invention include Acetobacter aceti, Acetobacter tropicalis, Acetobacter pasteurianus and the like.
Among these, Acetobacter aceti is preferable from the viewpoint that the obtained component has a higher ceramide and / or glucosylceramide production promoting effect.

Examples of microorganisms belonging to the genus Leuconostoc used in the present invention include Leuconostoc mesenteroides, Leuconostoc lactis, Leuconostoc gelidum, Leuconostoc carnosum, Leuconostoc fallax, Leuconostoc citreum, Leuconostoc argentinum, Leuconostoc pseudomesenteroides and the like.
Among these, Leuconostoc mesenteroides is preferable from the viewpoint that the obtained component has a higher ceramide and / or glucosylceramide production promoting effect.

  All of the microorganisms (bacteria) according to the present invention are the Chinese Common Microorganisms Storage Center (CGMCC) and the Chinese Traditional Culture Storage Center (CCTCC), the Chinese High School Industrial Microbial Resources and Information Center (CICIM: Culture & Information Center of Industrial) It can also be purchased through commercial routes such as the Microorganism of China Universities.

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

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

  The soybean residue used in the present invention is preferably obtained by immersing soybean in water, absorbing water, grinding and filtering, removing water, and drying. In the food industry, it is also possible to use a soybean residue, such as okara, which is left in the process of producing a soybean product in which soybean milk is extracted.

  Examples of the saccharide include sucrose, maltose, and sucrose. In addition, they can be used alone or in combination. Sucrose can be suitably used from the viewpoint of improving the effect of promoting ceramide and / or glucosylceramide production.

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

  From the viewpoint of enhancing the physiological characteristics of microorganisms with bacteria and the effect of promoting the production of ceramide and / or glucosylceramide, it is preferable to perform the culture at a culture temperature of 27 to 35 ° C.

  From the viewpoint of enhancing the physiological characteristics of microorganisms with bacteria and the effect of promoting production of ceramide and / or glucosylceramide, the cells are cultured for 1 to 7 days, more preferably 48 to 96 hours, and even more preferably 68 to 76 hours. Is preferred.

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

  The supernatant obtained by centrifugation may be sterilized by adding ethanol, and the ceramide and / or glucosylceramide production promoter of the present invention may be used.

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

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

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

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

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

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

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

  The blended amount of the ceramide and / or glucosylceramide production promoter of the present invention into the external preparation for the skin sufficiently promotes the synthesis of ceramide and / or glucosylceramide as a dried product of fermented extract of soybean residue (solid content conversion). The skin moisturizing ability is efficiently increased, and the color and odor of the culture are difficult to be produced. Therefore, the amount is preferably 0.01 to 20 w / v%, particularly preferably 0.1 to 10 w of the whole skin external preparation. / v%.

EXAMPLES Next, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to these.
All the microorganisms used in the examples were purchased from Gangnam University and entrusted to China High School Industrial Microbial Resources and Information Center CICIM.

(Example 1-1)
<Manufacture of ceramide and / or glucosylceramide production promoter>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.
Next, 100 ml of the above medium was placed in an Erlenmeyer flask, inoculated with Bacillus subtilis (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1677)) in a prescribed amount, and cultured at 30 ° C. for 3 days. .
The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 1-A and 1-B of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 1-A and 1-B are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.
Next, the medium containing no growth factor was changed in the 12-well plate, and the product of the present invention (samples 1-A and 1-B) and the control (50 v / v% ethanol solution) were added to the 12-well plate (n = 2). Added to. The cells were cultured for 3 days under the same conditions.
Thereafter, the culture medium of the supernatant was removed, and the cells were washed twice with PBS (2 mL / Well). Then, 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for analyzing the amount of ceramide, glucosylceramide and protein later.

(Confirmation of ceramide / glucosylceramide production promoting effect)
First, methanol: chloroform (2.5 ml: 1.25 ml) was added to the PBS solution containing the collected cells and stirred, and then shaken for 20 minutes. Thereafter, centrifugation (3000 rpm, 5 minutes) was performed, and the supernatant a and the precipitate b were separated. The supernatant a was subjected to the analysis of the amount of ceramide / glucosylceramide, and the precipitate b was subjected to the next protein amount analysis.
(1) Analysis of the amount of ceramide / glucosylceramide PBS: chloroform (1.25 ml: 1.25 ml) is added to the supernatant a, shaken for 20 minutes, centrifuged (3000 rpm, 5 minutes), and chloroform in the lower phase. The layer was isolated and collected. The recovered chloroform layer was dried at 30 ° C. in a nitrogen atmosphere. The dried product was dissolved in 100 μl of a chloroform-methanol mixed solvent (volume ratio = 2.5 ml: 1.25 ml) and subjected to quantification of the amount of ceramide and the amount of glucosylceramide by the following thin layer chromatography.
A ceramide (or glucosylceramide) standard product and a sample solution were placed in a dry TLC plate, and thin layer chromatography was performed with chloroform: methanol: acetic acid = 190: 9: 1 (v / v / v) as a developing solvent. . After the developing agent reached the top of the TCL plate, the developing agent was dried with a dryer. The above operation was then repeated once. Thereafter, thin layer chromatography was performed using chloroform: methanol: acetone = 76: 20: 4 (v / v / v) as a developing solvent. After the developing agent reached a position 2.5 cm from the bottom of the TCL plate, the development was stopped and the developing agent was dried with a dryer. A phosphoric acid solution of copper sulfate was blown and dried, and the TCL plate was placed on a 180 ° C. heater and heated for 7 minutes to develop the color. The developed TLC plate was scanned to analyze the amount of ceramide (or glucosylceramide) (Cer and GlyCer, respectively, unit μg / mL).

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

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

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

  As is apparent from Table 1, the effects of promoting the production of ceramide and glucosylceramide were observed in samples 1-A and 1-B of Example 1-1.

(Example 1-2)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 1-1, Bacillus subtilis (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1923) as a microorganism of the genus Bacillus In the same manner as in Example 1-1, except that the above was used.

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

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

  As is clear from Table 1, the ceramide and / or glucosylceramide production promoting effect was observed in Samples 1-C and 1-D of Example 1-2.

(Example 1-3)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 0051, Bacillus acidicola (Chinese High School Industrial Microbial Resources and Information Center (CICIM is numbered as CICIM B1297) is used as a microorganism belonging to the genus Bacillus. ) Was used in the same manner as in Example 1-1.

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

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

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

  As is clear from Table 1, the ceramide production promoting effect was observed in Samples 1-E and 1-G of Example 1-3.

(Example 1-4)

In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 1-1, Bacillus gibsonii (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1171) as a microorganism belonging to the genus Bacillus In the same manner as in Example 1-1, except that the above was used.

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

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

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

  As is clear from Table 1, the ceramide and / or glucosylceramide production promoting effect was observed in Samples 1-H, 1-I, and 1-J of Example 1-4.

(Example 1-5)

In <Production of ceramide and / or glucosylceramide production promoter> described in Example 1-1, Bacillus licheniformis (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1294) as a microorganism of the genus Bacillus In the same manner as in Example 1-1, except that the above was used.

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

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

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

  As is clear from Table 1, the ceramide and / or glucosylceramide production promoting effect was observed in samples 1-K, 1-L and 1-M of Example 1-5.

(Example 1-6)

In <Production of Ceramide and / or Glucosylceramide Production Accelerator> described in Example 1-1, Bacillus licheniformis (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1282) as a microorganism belonging to the genus Bacillus In the same manner as in Example 1-1, except that the above was used.

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

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

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

  As is clear from Table 1, the ceramide and / or glucosylceramide production promoting effect was observed in the samples 1-N, 1-O, and 1-P of Example 1-6.

(Example 1-7)

In <Production of Ceramide and / or Glucosylceramide Production Accelerator> described in Example 1-1, Bacillus licheniformis (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1298) as a microorganism belonging to the genus Bacillus In the same manner as in Example 1-1, except that the above was used.

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

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

  As is clear from Table 1, Samples 1-Q and 1-R of Example 1-7 were found to have a glucosylceramide production promoting effect.

Regarding samples 1-A to 1-R, the amount of ceramide and glucosylceramide were not added to the 12-well plate containing the activated human keratinocytes (ie, the cell culture was not performed). Quantity analysis was performed. Except for the above, the procedure was the same as in Example 1-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any of samples 1-A to 1-R.
From this, ceramide or glucosylceramide does not exist in the product itself of the present invention, but the product of the present invention has an effect of promoting ceramide and / or glucosylceramide production and is used as a ceramide and / or glucosylceramide production promoter. I understand that.

(Example 2-1)
<Manufacture of ceramide and / or glucosylceramide production promoter>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.
Next, 100 ml of the above medium was placed in an Erlenmeyer flask, inoculated with Lactobacillus brevis (China High School Industrial Microbial Resources and Information Center (consigned by CICIM as number CICIM B1257)) in a prescribed amount, and cultured at 30 ° C. for 3 days. .

The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH was added, and 1 ml of the mixture was used as sample 2-A-1 of the ceramide and / or glucosylceramide production promoter of the present invention in a refrigerator at 4 ° C. Store and subject to cell culture.
The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-A-2 and 2-A-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-A-2 and 2-A-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.

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

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

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

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

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

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

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

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

  As is clear from Table 2, it was confirmed that Samples 2-A-1, 2-A-2 and 2-A-3 of Example 2-1 exerted a glucosylceramide production promoting effect.

(Example 2-2)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus brevis (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1199) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-B-2 and 2-B-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-B-2 and 2-B-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

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

  As is clear from Table 2, it was confirmed that Samples 2-B-1, 2-B-2 and 2-B-3 of Example 2-2 exerted a glucosylceramide production promoting effect.

(Example 2-3)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus farciminis (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1173) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-C-2 and 2-C-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-C-2 and 2-C-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

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

  As is clear from Table 3, it was confirmed that Samples 2-C-1, 2-C-2 and 2-C-3 of Example 2-3 exerted a ceramide production promoting effect.

(Example 2-4)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus pentosus (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1174) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-D-2 and 2-D-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-D-2 and 2-D-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-D-1, 2-D-2, and 2-D-3 was evaluated. The results are shown in Table 4.

  As apparent from Table 4, it was confirmed that Samples 2-D-1, 2-D-2, and 2-D-3 of Example 2-4 exerted a ceramide production promoting effect.

(Example 2-5)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1172) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-E-2 and 2-E-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-E-2 and 2-E-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-E-1, 2-E-2, 2-E-3 was evaluated. The results are shown in Table 5.

  As is clear from Table 5, it was confirmed that Samples 2-E-1, 2-E-2, 2-E-3 of Example 2-5 exerted a glucosylceramide production promoting effect.

(Example 2-6)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1189) The culture was carried out in the same manner as in Example 2-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-F-1,2-F-2 of the ceramide and / or glucosylceramide production accelerator of the present invention, and each of samples 2-F-1,2-F-2 was 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of sample 2-F-1,2-F-2 was evaluated. The results are shown in Table 5.

  As is apparent from Table 5, it was confirmed that Samples 2-F-1,2-F-2 of Example 2-6 exerted the effect of promoting ceramide and glucosylceramide production.

(Example 2-7)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1187) The culture was carried out in the same manner as in Example 2-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as sample 2-G-1 of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 2-G-1 is dissolved in 50 v / v% EtOH to obtain 1 v / v% and 0.2%. After preparing a solution with a concentration of 1 v / v%, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-G-1 was evaluated. The results are shown in Table 5.

  As apparent from Table 5, it was confirmed that Sample 2-G-1 of Example 2-7 fulfilled the effect of promoting production of ceramide and glucosylceramide.

(Example 2-8)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1170) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-H-2 and 2-H-3 of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 2-H-2 and 2-H-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of the samples 2-H-1, 2-H-2, and 2-H-3 was evaluated. The results are shown in Table 5.

  As is clear from Table 5, it was confirmed that Samples 2-H-1, 2-H-2 and 2-H-3 of Example 2-8 exerted a ceramide and / or glucosylceramide production promoting effect. .

(Example 2-9)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1188) The culture was carried out in the same manner as in Example 2-1.

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

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

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-I-1, 2-I-2 was evaluated. The results are shown in Table 5.

  As is clear from Table 5, it was confirmed that Samples 2-I-1, 2-I-2 of Example 2-9 exerted a glucosylceramide production promoting effect.

(Example 2-10)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus coryniformis (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1243) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-J-2 and 2-J-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-I-2 and 2-J-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-J-1, 2-J-2, and 2-J-3 was evaluated. The results are shown in Table 6.

  As is apparent from Table 6, it was confirmed that Samples 2-J-1, 2-J-2, 2-J-3 of Example 2-10 exerted ceramide and / or glucosylceramide production promoting effect. .

(Example 2-11)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus coryniformis (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1190) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-K-2 and 2-K-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-K-2 and 2-K-3 were each converted to 50 v / After dissolving in v% EtOH to prepare solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-K-1, 2-K-2, and 2-K-3 was evaluated. The results are shown in Table 6.

  As is clear from Table 6, it was confirmed that Sample 2-K-1 of Example 2-11 exerted a glucosylceramide production promoting effect.

(Example 2-12)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus coryniformis (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1259) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-L-2 and 2-L-3 of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 2-L-2 and 2-L-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-L-1, 2-L-2 and 2-L-3 was evaluated. The results are shown in Table 6.

  As is clear from Table 6, it was confirmed that the samples 6L-2 and 6L-3 of Example 612 exerted a glucosylceramide production promoting effect.

(Example 2-13)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus curvatus (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1248) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 2-M-2 and 2-M-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-M-2 and 2-M-3 are each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of samples 2-M-1, 2-M-2, and 2-M-3 was evaluated. The results are shown in Table 7.

  As is clear from Table 7, it was confirmed that Samples 2-M-1,2-M-3 of Example 2-13 exerted ceramide and / or glucosylceramide production promoting effect.

(Example 2-14)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus curvatus (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1413) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-N-2 and 2-N-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-N-2 and 2-N-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-N-1,2-N-2, 2-N-3 was evaluated. The results are shown in Table 7.

  As is clear from Table 7, it was confirmed that Sample 2-N-2 of Example 2-14 fulfilled the effect of promoting production of ceramide and glucosylceramide.

(Example 2-15)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1430) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-O-2 and 2-O-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-O-2 and 2-O-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-O-1,2-O-2, 2-O-3 was evaluated. The results are shown in Table 8.

  As is apparent from Table 8, it was confirmed that the samples 2-O-1, 2-O-2 and 2-O-3 of Example 2-15 exerted the ceramide and / or glucosylceramide production promoting effect. .

(Example 2-16)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1441) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-P-2 and 2-P-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-P-2 and 2-P-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-P-1,2-P-2, 2-P-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-P-1, 2-P-2, 2-P-3 of Example 2-16 exerted a ceramide and / or glucosylceramide production promoting effect. .

(Example 2-17)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1191) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-Q-2 and 2-Q-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-Q-2 and 2-Q-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-Q-1, 2-Q-2, and 2-Q-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-Q-1, 2-Q-2 and 2-Q-3 of Example 2-17 exerted a ceramide and / or glucosylceramide production promoting effect. .

(Example 2-18)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1179) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-R-2 and 2-R-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-R-2 and 2-R-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of the samples 2-R-1, 2-R-2 and 2-R-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-R-1, 2-R-2 and 2-R-3 of Example 2-18 exerted a glucosylceramide production promoting effect.

(Example 2-19)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1249) The culture was carried out in the same manner as in Example 2-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-S-1,2-S-2 of the ceramide and / or glucosylceramide production promoter of the present invention, and each of samples 2-S-1,2-S-2 was 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-S-1,2-S-2 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-S-1,2-S-2 of Example 2-19 exerted a glucosylceramide production promoting effect.

(Example 2-20)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1250) The culture was carried out in the same manner as in Example 2-1.

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

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

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

  As is clear from Table 8, it was confirmed that Sample 2-T-1,2-T-2 of Example 2-20 exerted a glucosylceramide production promoting effect.

(Example 2-21)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1255) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as samples 2-U-2 and 2-U-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-U-2 and 2-U-3 are each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-U-1, 2-U-2 and 2-U-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-U-1, 2-U-2 and 2-U-3 of Example 2-21 exerted a glucosylceramide production promoting effect.

(Example 2-22)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1433) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-V-2 and 2-V-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-V-2 and 2-V-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-V-1, 2-V-2, 2-V-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-V-1, 2-V-2 and 2-V-3 of Example 2-22 exerted a glucosylceramide production promoting effect.

(Example 2-23)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1432) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-W-2 and 2-W-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-W-2 and 2-W-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-W-1, 2-W-2, 2-W-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-W-1, 2-W-2 and 2-W-3 of Example 2-23 exerted ceramide and glucosylceramide production promoting effects.

(Example 2-24)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus plantarum (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1442) The culture was carried out in the same manner as in Example 2-1.

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

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-X-2 and 2-X-3 of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 2-X-2 and 2-X-3 were each 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of samples 2-X-1, 2-X-2, and 2-X-3 was evaluated. The results are shown in Table 8.

  As is clear from Table 8, it was confirmed that Samples 2-X-1, 2-X-2, and 2-X-3 of Example 2-24 had an effect of promoting ceramide and glucosylceramide production.

(Example 2-25)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus alimentarius (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1439) The culture was carried out in the same manner as in Example 2-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as samples 2-Y-1,2-Y-2 of the ceramide and / or glucosylceramide production accelerator of the present invention, and each of samples 2-Y-1,2-Y-2 was 50 v / v. After dissolving in% EtOH and preparing solutions with concentrations of 1 v / v% and 0.1 v / v%, they were stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Samples 2-Y-1,2-Y-2 was evaluated. The results are shown in Table 9.

As is clear from Table 9, it was confirmed that Sample 2-Y-1,2-Y-2 of Example 2-25 fulfilled the effect of promoting ceramide and / or glucosylceramide production.

(Example 2-26)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism of the genus Lactobacillus, Lactobacillus alimentarius (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1440) The culture was carried out in the same manner as in Example 2-1.

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

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-Z-1 was evaluated. The results are shown in Table 9.

  As is clear from Table 9, it was confirmed that Sample 2-Z-1 of Example 2-26 exerted a ceramide production promoting effect.

(Example 2-27)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 2-1, as a microorganism belonging to the genus Lactobacillus, Lactobacillus farciminis (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1443) The culture was carried out in the same manner as in Example 2-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. The 2.5-fold volume of EtOH was added to the remaining culture supernatant, and the mixed solution to which the EtOH was added was treated for 10 minutes with an ultrasonic treatment apparatus, and the supernatant was collected by centrifugation at 3000 rpm for 30 mins. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was designated as sample 2-a-1 of the ceramide and / or glucosylceramide production accelerator of the present invention, and sample 2-a-1 was further dissolved in 50 v / v% EtOH to obtain 0.1 v / v%. After preparing a solution of a concentration, it was stored in a refrigerator at 4 ° C. and used for the next cell culture.

  In the same manner as in Example 2-1, the ceramide / glucosylceramide production promoting effect of Sample 2-a-1 was evaluated. The results are shown in Table 9.

  As is clear from Table 9, it was confirmed that Sample 2-a-1 of Example 2-27 fulfilled the effect of promoting ceramide production.

For each of the above samples, the amount of ceramide and the amount of glucosylceramide were analyzed without adding it to the 12-well plate containing the activated human keratinocytes (that is, without performing the cell culture). went. Except for the above, the procedure was the same as in Example 2-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any one of the above samples.
From this, ceramide or glucosylceramide does not exist in the product itself of the present invention, but the product of the present invention has an effect of promoting ceramide and / or glucosylceramide production and is used as a ceramide and / or glucosylceramide production promoter. I understand that.

(Example 3-1)
<Manufacture of ceramide and / or glucosylceramide production promoter>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.
Next, 100 ml of the above medium was placed in an Erlenmeyer flask, inoculated with Acetobacter aceti (Chinese High School Industrial Microbial Resources and Information Center (consigned by CICIM as number CICIM B0137)) in a prescribed amount, and cultured at 30 ° C. for 3 days. .
The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixture is used as sample 3-A of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.
The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 3-B and 3-C of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 3-B and 3-C were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

<Evaluation of ceramide and / or glucosylceramide production promoter>

(Cell culture)
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.
Next, the medium containing no growth factor was changed in the 12 well plate, and the product of the present invention (samples 3-A, 3-B, 3-C) and control (50 v / v% ethanol solution) were added to the 12 well plate ( n = 2). The cells were cultured for 3 days under the same conditions.
Thereafter, the culture medium of the supernatant was removed, and the cells were washed twice with PBS (2 mL / Well). Then, 1 mL PBS was added, and each cell was collected in a test tube with a cell harvester. This was used as a sample for analyzing the amount of ceramide, glucosylceramide and protein later.

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

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

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

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

(3) Results of Analysis Cer / Pro and GlyCer / Pro were measured for each of the product of the present invention and the control sample as shown in the following formula. The relative values of Cer / Pro and GlyCer / Pro of the present invention were calculated and shown in Table 10 when each of Cer / Pro and GlyCer / Pro of the control was set to 1. In addition, the relative value (Pro. (%)) Of the protein amount per well of the present invention when the amount of protein per well of the control is 100 was calculated, whereby the amount of ceramide and glucosyl per well were calculated. The amount of ceramide (relative to the control) was determined. The results are also shown in Table 10.
Cer / Well = Cer / Pro × Pro. (%)
GlyCer / Well = GlyCer / Pro × Pro. (%)

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

  As apparent from Table 10, the samples 3-A, 3-B, and 3-C of Example 3-1 were found to have a glucosylceramide production promoting effect.

(Example 3-2)
In <Production of Ceramide and / or Glucosylceramide Production Accelerator> described in Example 3-1, Acetobacter aceti (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B0142) as a microorganism belonging to the genus Acetobacter The culture was performed in the same manner as in Example 3-1.

The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixture is used as sample 3-D of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.
The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is designated as ceramide and / or glucosylceramide production promoters 3-E and 3-F of the present invention, and samples 3-E and 3-F are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 3-1, the ceramide / glucosylceramide production promoting effect of Samples 3-D, 3-E, and 3-F was evaluated. The results are shown in Table 10.

  As is clear from Table 10, Samples 3-D, 3-E, and 3-F of Example 3-2 were found to have an effect of promoting production of ceramide and glucosylceramide.

(Example 3-3)
In <Production of Ceramide and / or Glucosylceramide Production Accelerator> described in Example 3-1, Acetobacter aceti (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B0145) as a microorganism belonging to the genus Acetobacter The culture was performed in the same manner as in Example 3-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixture is used as sample 3-G of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 3-H and 3-I of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 3-H and 3-I were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 3-1, the ceramide / glucosylceramide production promoting effect of Samples 3-G, 3-H, and 3-I was evaluated. The results are shown in Table 10.

  As is apparent from Table 10, Samples 3-G, 3-H, and 3-I of Example 3-3 were found to have a ceramide production promoting effect.

(Example 3-4)
In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 3-1, Acetobacter aceti (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B0133) as a microorganism belonging to the genus Acetobacter The culture was performed in the same manner as in Example 3-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixture is used as sample 3-J of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is used as samples 3-K and 3-L of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 3-K and 3-L are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 3-1, the ceramide / glucosylceramide production promoting effect of Samples 3-J, 3-K, and 3-L was evaluated. The results are shown in Table 10.

  As is clear from Table 10, the ceramide production promoting effect was observed in the samples 3-J, 3-K, and 3-L of Example 3-4.

(Example 3-5)

In <Production of Ceramide and / or Glucosylceramide Production Accelerator> described in Example 3-1, Acetobacter aceti (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B0128) as a microorganism of the genus Acetobacter The culture was performed in the same manner as in Example 3-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixed solution is used as sample 3-M of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is used as samples 3-N and 3-O of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 3-N and 3-O are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 3-1, the ceramide / glucosylceramide production promoting effect of Samples 3-M, 3-N, and 3-O was evaluated. The results are shown in Table 10.

  As apparent from Table 10, the samples 3-M, 3-N and 3-O of Example 3-5 were found to have a ceramide production promoting effect.

(Example 3-6)

In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 3-1, Acetobacter aceti (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B0130) as a microorganism of the genus Acetobacter The culture was performed in the same manner as in Example 3-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times volume of EtOH is added, and 1 ml of the mixture is used as sample 3-P of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product was used as samples 3-Q and 3-R of the ceramide and / or glucosylceramide production accelerator of the present invention, and samples 3-Q and 3-R were dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 3-1, the ceramide / glucosylceramide production promoting effect of the samples 3-P, 3-Q, and 3-R was evaluated. The results are shown in Table 10.

  As is clear from Table 10, the ceramide production promoting effect was observed in the samples 3-P, 3-Q, 3-R of Example 3-6.

(Example 3-7)

In <Manufacture of ceramide and / or glucosylceramide production promoter> described in Example 3-1, Acetobacter aceti (Chinese High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B0120) as a microorganism of the genus Acetobacter The culture was performed in the same manner as in Example 3-1.

  The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixture is used as sample 3-S of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.

  The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is used as samples 3-T and 3-U of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 3-T and 3-U are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.

  In the same manner as in Example 3-1, the ceramide / glucosylceramide production promoting effect of Samples 3-S, 3-T, and 3-U was evaluated. The results are shown in Table 10.

  As apparent from Table 10, the samples 3-S and 3-T of Example 3-7 were found to have a glucosylceramide production promoting effect.

  For samples 3-A to 3-U, the amount of ceramide and glucosylceramide were not added to the 12-well plate containing the activated human keratinocytes (ie, the cell culture was not performed). Quantity analysis was performed. Except for the above, the procedure was the same as in Example 3-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any of samples 3-A to 3-U.

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

(Example 4-1)
<Manufacture of ceramide and / or glucosylceramide production promoter>
First, a medium was prepared by the following method. Soybeans were placed in water and impregnated for 12 hours to allow the soybeans to absorb water sufficiently. The soybean which absorbed water was ground and pulverized, filtered and dripped and dried to obtain soybean residue. The soybean residue was mixed with glucose and water so that the concentration of soybean residue was 10 w / v% and the concentration of glucose was 1 w / v% to obtain a medium.

  Next, 100 ml of the above medium was placed in an Erlenmeyer flask, inoculated with Leuconostoc mesenteroides (China High School Industrial Microbial Resources and Information Center (consigned by CICIM as number CICIM B1254)) in a prescribed amount, and cultured at 30 ° C. for 3 days. .

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

<Evaluation of ceramide and / or glucosylceramide production promoter>
(Cell culture)
First, normal human activated keratinocytes (Cascade Co., Ltd) are used as primary cells in a 75 cm ² culture flask (2 ml of medium containing growth factors) in an incubator at 7 ° C. and 5% CO ₂ . The cells were cultured until reaching a state of ˜90% confluence. Passage cells were continuously cultured in a 175 cm ² culture flask until reaching 80-90% confluence. Subcultured human epidermal keratinocytes are seeded at 2 mL each in a 12-well plate (1 × 10 ⁵ cells / mL) and brought to 80-90% confluence in a 37 ° C., 5% CO ₂ incubator. Continued culture until reached.

  Next, the medium containing no growth factor in the 12-well plate was changed, and the product of the present invention (samples 4-A, 4-B, 4-C) and control (50 v / v% ethanol solution) were added to the 12-well plate ( n = 2). The cells were cultured for 3 days under the same conditions.

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

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

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

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

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

(3) Results of analysis Cer / Pro was measured for each of the product of the present invention and the control sample as shown below. Table 11 shows the relative values of Cer / Pro of the present invention when Cer / Pro of the control is 1. Further, the relative value (Pro. (%)) Of the protein amount per well of the present invention when the amount of protein per well of the control is 100 was calculated, and thereby the amount of ceramide per control (control) Relative amount). The results are also shown in Table 11.
Cer / Well = Cer / Pro × Pro. (%)

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

  As is clear from Table 11, the ceramide production promoting effect was observed in Samples 4-A, 4-B and 4-C of Example 4-1.

(Example 4-2)
In <Production of ceramide and / or glucosylceramide production promoter> described in Example 4-1, Leuconostoc mesenteroides (China High School Industrial Microbial Resources and Information Center (consigned to CICIM as number CICIM B1364) as microorganisms of the genus Leuconostoc The culture was performed in the same manner as in Example 4-1.
The obtained culture solution was centrifuged at 3000 rpm for 10 minutes. To the remaining culture supernatant, 2.5 times the volume of EtOH is added, and 1 ml of the mixture is used as sample 4-D of the ceramide and / or glucosylceramide production promoter of the present invention and stored in a refrigerator at 4 ° C. And subject to cell culture.
The mixed solution to which EtOH was added was treated with an ultrasonic treatment apparatus for 10 minutes, and the supernatant was collected by centrifugation at 3000 rpm for 30 minutes. The supernatant (about 70 mL) was filtered with a filter paper, and the filtrate was further dried with a vacuum dryer to a constant weight. The dried product is used as samples 4-E and 4-F of the ceramide and / or glucosylceramide production promoter of the present invention, and samples 4-E and 4-F are dissolved in 50 v / v% EtOH, respectively. After preparing a solution with a concentration of v% and 0.1 v / v%, it was stored in a refrigerator at 4 ° C. and subjected to the next cell culture.
In the same manner as in Example 4-1, the ceramide production promoting effect of Samples 4-D, 4-E, and 4-F was evaluated. The results are shown in Table 11.
As is clear from Table 11, the ceramide production promoting effect was observed in Samples 4-D, 4-E, and 4-F of Example 4-2.

  For samples 4-A to 4-F, the amount of ceramide and glucosylceramide were not added to the 12-well plate containing the human activated keratinocytes (ie, the cell culture was not performed). Quantity analysis was performed. Except for the above, the procedure was the same as in Example 4-1. As a result, it was confirmed that ceramide or glucosylceramide was not present in any of samples 4-A to 4-F.

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

  The ceramide and / or glucosylceramide production promoter of the present invention can be used for pharmaceuticals, quasi drugs, cosmetics and the like.

Claims (10)

Bacillus licheniformis, Lactobacillus brevis, Lactobacillus pentosu s, L euconostoc mesenteroides, Bacillus acidicola, Bacillus gibsonii, Lactobacillus farciminis, Lactobacillus coryniformis, Lactobacillus curvatus, Lactobacillus alimentarius, and Acetobacter ACET i or al 1 or more microorganisms soybean residue and saccharides selected A method for producing a ceramide and / or glucosylceramide production promoter, wherein the ceramide and / or glucosylceramide production promoter is collected from the culture supernatant.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 1, wherein the saccharide is sucrose.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 1, wherein the medium comprises soybean residue, sucrose and water.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 3, wherein the soybean residue is contained in the medium at 8 to 12 w / v% and the saccharide at 0.8 to 1.2 w / v%. .   The method for producing a ceramide and / or glucosylceramide production promoter according to any one of claims 1 to 4, wherein the culture temperature is 27 to 35 ° C.   The method for producing a ceramide and / or glucosylceramide production promoter according to any one of claims 1 to 5, wherein the culture time is 1 to 7 days.   The method for producing a ceramide and / or glucosylceramide production promoter according to any one of claims 1 to 6, wherein the supernatant is obtained by centrifuging the culture solution obtained by the culture.   The method for producing a ceramide and / or glucosylceramide production promoter according to claim 7, wherein ethanol is added to the centrifuged supernatant to obtain a ceramide and / or glucosylceramide production promoter.   The ceramide and / or glucosyl according to claim 8, which is obtained by further purifying the ethanol-added product by sonication, centrifugation, filtration, or vacuum drying to obtain a ceramide and / or glucosylceramide production promoter. A method for producing a ceramide production promoter.   A ceramide and / or glucosylceramide production promoter obtained by the production method according to claim 1.