CN102465150B - Manufacturing method of ceramide generation accelerator - Google Patents
Manufacturing method of ceramide generation accelerator Download PDFInfo
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- CN102465150B CN102465150B CN201110289055.5A CN201110289055A CN102465150B CN 102465150 B CN102465150 B CN 102465150B CN 201110289055 A CN201110289055 A CN 201110289055A CN 102465150 B CN102465150 B CN 102465150B
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Abstract
The invention provides a manufacturing method of a ceramide generation accelerator and/or a glucosylceramide generation accelerator, wherein Pichia microorganisms are cultured in a culture medium containing beam dregs and saccharides, and the ceramide generation accelerator and/or the glucosylceramide generation accelerator are/is obtained from an obtained supernatant. According to the manufacturing method disclosed by the invention, by utilization of easily obtainable substances as the culture medium and specific microorganisms, the ceramide generation accelerator and/or the glucosylceramide generation accelerator can be acquired at low cost and high efficiency.
Description
Technical field
The present invention relates to promote skin to produce ceramide and/or the ceramide production accelerant of glucose ceramide and/or the manufacture method of glucose ceramide production accelerant.
Background technology
Skin has the ability of generation ceramide (ceramide) and glucose ceramide (glucosylceramide).In skin, particularly assembling the ceramide being obtained by epidermic cell synthesis secretion in horny layer of epidermis, it is the main component of intercellular lipid.And be temporarily stored with the form of glucose ceramide or sphingomyelin (sphingomyelin) by the synthetic ceramide obtaining of epidermic cell; Be discharged to afterwards extracellular, under the effect of glucose cerebrosidase (glucocerebrosidase) and sphingomyelinase (sphingomyelinase), again form ceramide, thereby performance is as the function of intercellular lipid.Ceramide in skin and glucose ceramide have the physiological action that improves the moisture-keeping functions of skin and the barrier function of skin etc.
But recently, due to the impact of environment, weather etc., it is dry that skin easily becomes, therefore produce by skin self moisture-keeping functions that ceramide etc. improves skin and just seem not enough.
Thereby people have attempted supplementing to skin from outside the method for ceramide.For example, in patent documentation 1, record microorganism that cultivation candiyeast (Candida) belongs to and manufactured the method for glucose ceramide, by this glucose ceramide being coated on to the moisture-keeping functions that can improve skin on the drying property skin of human body.The method is directly from the thalline cultivation, to obtain the glucose ceramide of a large amount of existence, therefore the ceramide skeleton of the glucose ceramide of synthesized is different with the ceramide skeleton of the ceramide type existing in human body, and it has carbon-carbon double bond and have side chain at Liang Chu.Therefore, this glucose ceramide obtaining that directly extracts from microorganism is coated on human body skin, likely can not adapts with human body skin and produce the problem of safety in utilization aspect.
In addition, from outside to the same inadequate problem of persistence that has humidity-holding effect such as the method for the supplementary ceramide of skin and the wetting Agent for Printing Inks of use in the past, and the skin of some state is insufficient to the absorption of ceramide from extraneous supply etc., thereby cause giving full play to humidity-holding effect.
And on the other hand, if can strengthen or promote the generation ability of ceramide or the glucose ceramide of skin itself, without from external complement ceramide or glucose ceramide, and can improve by promoting skin self to produce ceramide and/or glucose ceramide barrier function and the moisture-keeping functions of skin.Like this, because being produces ceramide or glucose ceramide by skin self, synthesized be the ceramide etc. of the conventional structure that has of skin, thereby there is not the safety issue bad etc. with the adaptability of skin in it.
Therefore recently quite popular about the research of material that produces the generation that promotes epidermis ceramide etc. in keratoderma.Wherein, as the material that can promote that ceramide generates, that has reported has a ceramide production accelerant (patent documentation 2～4) take the extract of glossy ganoderma, bee pollen (Bee Pollen), Ligusticum wallichii etc. as effective constituent.But because these ceramide production accelerants are all to extract and obtain from plant material, it extracts and operates the length that expends time in, and restricted aspect the utilizability of natural resource, thereby its manufacturing cost is high.
In addition, in patent documentation 5, a kind of synthetic ceramide synthesis accelerators of ceramide of the epidermic cell self that can activate skin surface inside is disclosed, it is take the bacterium culture that contains nicotinic acid and/or niacinamide as effective constituent, and this bacterium culture can be lactic acid bacteria culture, bifidobacterium culture, mushroom thalline culture or Yeast culture.But the substratum using in the preparation process of this promotor is in fact using nicotinic acid and/or niacinamide as effective constituent, therefore the preparation cost of this ceramide synthesis accelerators is higher.
In addition, finish red (Pichia) genus yeast and there are following character and purposes etc.
Pichia bacterium cell is spherical in shape, oval, elongation shape, tapered once in a while, but does not form pinnacle.Vegetative propagation mode is polygon budding, and some kind can form arthrospore.Each ascus comprises 1～4 thecaspore conventionally, once in a while more than 4.Spore is cap shape, semisphere or spherically has a rib.Ascus maturation is generally split afterwards, and minority is not split, and engages or do not engage to form ascus, is bonded between parent cell and sprout or two independent cells and occurs.Mycelia and pseudohypha all can be used as ascus, but form can not become large or become fusiform, of the same clan or heterothally; Can assimilate nitrate, DBB reaction negative.
The classification position of finishing red (Pichia) genus yeast is: Ascomycota (Ascomycota), Hemiascomycetes (Hemiascomycetes), Saccharomycetes (Saccharomycetales), Saccharomycetaceae (Saccharomycetaceae), Pichia (Pichia).
The purposes that finishes red (Pichia) genus yeast has, and for example, utilizes methyl alcohol to produce some useful matteies, as microbiotic, and bioprotein etc.; Or also can be used as genetic engineering bacterium uses.Pichia pastoris phaff, for the production of reduced glutathion, also has other some pichia spp for the production of some protein/enzyme classes.
But, also do not utilize Pichia pastoris yeast to prepare the report of ceramide production accelerant or glucose ceramide production accelerant at present.
Patent documentation 1: TOHKEMY 2010-22217 communique
Patent documentation 2: TOHKEMY 2005-194240 communique
Patent documentation 3: TOHKEMY 2010-70499 communique
Patent documentation 4: TOHKEMY 2010-150237 communique
Patent documentation 5: Japanese kokai publication hei 9-194383 communique
Summary of the invention
The present invention makes in order to solve the technical problem existing in above-mentioned prior art, and object is to provide a kind of low cost and manufactures efficiently ceramide production accelerant and/or the method for glucose ceramide production accelerant.
The inventor has carried out wholwe-hearted research in order to solve the problems of the technologies described above, found that: in the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Pichia (Pichia), can obtain efficiently ceramide production accelerant and/or glucose ceramide production accelerant; Thereby complete the present invention.
The invention provides the manufacture method of a kind of ceramide production accelerant and/or glucose ceramide production accelerant, wherein, in the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Pichia (Pichia), in clear liquid, obtain from it ceramide production accelerant and/or glucose ceramide production accelerant.
Manufacturing method according to the invention, can use specific microorganism and utilize the material of the so easy acquisition of bean dregs as the main raw material of substratum, thereby can be cheap and obtain efficiently ceramide production accelerant and/or glucose ceramide production accelerant, this promotor can effectively be improved the ability of generation ceramide/glucose ceramide that skin has, thereby effectively improves the moisture-retaining capacity of skin.
Embodiment
Manufacture method of the present invention is in the substratum that contains bean dregs and carbohydrate, to cultivate the microorganism of Pichia, and in clear liquid, obtains ceramide production accelerant and/or glucose ceramide production accelerant from it.
As the microorganism of Pichia (Pichia) using in the present invention, can list not pichia spp (Pichia Kluyveri) etc. of abnormal pichia spp (Pichia anomala) conventional in field of food industry, Pichia guilliermondii (Pichia guilliermondii), Norway's pichia spp (Pichia norvegensis), film uncut jade pichia spp (Pichia membranifaciens), Christian Breton pichia spp (Pichia burtonii), pichia farinose (Pichia farinose), Crewe.
Wherein, the viewpoint from obtained product with higher ceramide generation facilitation effect and/or glucose ceramide generation facilitation effect is considered, preferably abnormal pichia spp (Pichiaanomala), Pichia guilliermondii (Pichia guilliermondii), Norway's pichia spp (Pichia norvegensis), film uncut jade pichia spp (Pichia membranifaciens), Christian Breton pichia spp (Pichia burtonii).
Wherein, film uncut jade pichia spp (Pichia membranifaciens) is called again Pichia membranaefaciens (Pichia membranaefaciens), the pichia spp of being addicted to drink (Pichia alcoholophila), bright spore pichia spp (Pichia hyalospora), Pichia scaptomyzae, Willia belgica or Zygopichia guilliermondii.
Christian Breton pichia spp (Pichia burtonii) is called again fiber candiyeast (Candidafibrae), Dematium chodati, Ke Dashi endomycopsi.sp (Endomycopsis chodati), hidden Christian Breton pichia spp (Hyphopichia burtonii) or shellfish thunder trichosporon (Trichosporonbehrendii).
The microorganism of Pichia (Pichia) involved in the present invention can be bought and obtain from commercial channel such as Chinese common micro-organisms preservation administrative center (CGMCC), Chinese Typical Representative culture collection center (CCTCC), Chinese Universities ' industrial microorganism resource and information centers (CICIM:Culture & InformationCenter of Industrial Microorganism of China Universities).
In manufacture method of the present invention, can use separately a kind of mentioned microorganism, also can combine mentioned microorganism of more than two kinds and use.
The substratum using in the present invention is the substratum that contains bean dregs and carbohydrate.Bean dregs and carbohydrate are all the materials easily obtaining, thereby substratum of the present invention has advantages of that cost is low.And, in addition, also can suitably add for example 1% peptone, 0.5% yeast powder etc.
Wherein, bean dregs refer to, pulverize after soybeans soaking being made in water it fully absorb moisture, more after filtration, the material that dewaters, drain and obtain.Also can use residual Soybeanresidue in the bean product manufacturing process of squeezing soya-bean milk etc. in food industries.
Carbohydrate can be glucose, sucrose, maltose etc., and they can use separately arbitrarily or mix use.And the viewpoint that produces facilitation effect and glucose ceramide generation facilitation effect from improving ceramide is considered, wherein preferred glucose.
The viewpoint that produces facilitation effect and glucose ceramide generation facilitation effect from improving ceramide is considered, medium optimization of the present invention is made up of bean dregs, glucose and water, further in preferred culture medium, the content of bean dregs is 5～20w/v%, more preferably 8～12w/v%, and the content of glucose is 0.2～2w/v%, more preferably 0.8～1.2w/v%.
Above-mentioned cultivation is preferably carried out in the viewpoint consideration that produces facilitation effect and glucose ceramide generation facilitation effect from physiological property and the raising ceramide of Pichia (Pichia) microorganism at 27～35 ℃.
The viewpoint consideration that produces facilitation effect and glucose ceramide generation facilitation effect from physiological property and the raising ceramide of Pichia (Pichia) microorganism, above-mentioned incubation time is preferably 1～7 day, more preferably 48～96 hours, more preferably 68～76 hours.
From obtaining the viewpoint of purer product, thereby preferably the culture obtaining by described cultivation is further refined and obtained supernatant liquor.As refining method, the appropriately combined conventional operation with filtration, centrifugation, ion-exchange or adsorption chromatography, solvent extraction, crystallization etc. is carried out as required.Preferably obtain supernatant liquor by centrifugation, the speed of this centrifugation is preferably 2000～4000rpm, 2500～3500rpm more preferably, and centrifugation time is preferably 0～20 minute, more preferably 5～15 minutes.
Can in the above-mentioned supernatant liquor of centrifugal rear acquisition, add ethanol, be used for carrying out sterilizing, and will add the supernatant liquor of ethanol as ceramide production accelerant and/or glucose ceramide production accelerant.
Also can from nutrient solution, remove by centrifugation thalline, then ultrasonication, centrifugation, reclaims supernatant liquor.Then, then by reclaim after supernatant liquid filtering remove impurity etc., under vacuum after drying treatment as ceramide of the present invention and/or glucose ceramide production accelerant.
By the Soybeanresidue fermented product extract of cultivating Pichia of the present invention (Pichia) microorganism and obtaining, there is the effect that makes the amount of ceramide and/or glucose ceramide increase in normal human's keratinocyte from culture supernatant.
Therefore, by the Soybeanresidue fermented product extract of cultivating Pichia of the present invention (Pichia) microorganism and obtaining from culture supernatant, can be for promoting ceramide and/or the therapeutic purpose of glucose ceramide generation or the use of non-therapeutic purpose, the ceramide production accelerant and/or the glucose ceramide production accelerant that also can be used as therapeutic purpose or non-therapeutic purpose use.The use of non-therapeutic purpose is that to improve looks be the use of object, or for maintaining the use etc. of state of health.And the use of described therapeutic purpose and the use of non-therapeutic purpose can completely be carried out with distinguishing.
By the Soybeanresidue fermented product extract of cultivating Pichia of the present invention (Pichia) microorganism and obtaining from culture supernatant, also can be for the manufacture of ceramide production accelerant and/or glucose ceramide production accelerant.
Ceramide production accelerant of the present invention and/or glucose ceramide production accelerant can be used as the ceramide that makes in stratum corneum and/or glucose ceramide to be increased and recovers and improve the uses such as the barrier function of skin and the pharmaceuticals of moisture-keeping functions, accurate pharmaceuticals, makeup.In addition, this ceramide production accelerant or glucose ceramide production accelerant also can be used as take ceramide generation promotion or glucose ceramide and produce promotion as accurate pharmaceuticals concept, that also indicate as required this concept, makeup use.
As the use form of ceramide production accelerant of the present invention and/or glucose ceramide production accelerant, can be the additive for adding to culturing cell, can be to be also coupled in the external composition for skin such as skin cleaning agent, makeup to use.For example, can enumerate the various forms such as astringent, emulsion, gel, frost, ointment.And as the base of external preparation, as long as known external application base, be not particularly limited.In the time of the various external composition for skin of preparation, can use individually ceramide production accelerant of the present invention and/or glucose ceramide production accelerant, or with appropriately combined use the such as the oiliness composition conventionally coordinating in the external composition for skin such as skin cleaning agent and makeup, wetting Agent for Printing Inks, powder, pigment, emulsifying agent, solubilizing agent, UV light absorber, thickening material, effective component, spices, fungi-proofing mould inhibitor, plant milk extract, alcohols.
Ceramide production accelerant of the present invention and/or glucose ceramide production accelerant are added in the epidermic cell of cultivation and promote in the situation of ceramide and/or glucose ceramide, addition as the dry thing of this promotor is preferably 0.0001～10w/v%, more preferably 0.001～5w/v%.When addition is 0.0001w/v% when above, can obtain sufficient facilitation effect; And when addition be below 10w/v% time, less to the pungency of epidermic cell.
When ceramide production accelerant of the present invention and/or glucose ceramide production accelerant are engaged in to external composition for skin and use, thereby effectively improve the moisture-retaining capacity of skin and be difficult for demonstrating the color of culture and the viewpoint of smell from the generation of effective promotion ceramide and/or glucose ceramide, its use level is preferably 0.01～20w/v% of external composition for skin total amount, more preferably 0.1～10w/v%.
Embodiment
Below, based on embodiment, the present invention will be described in more detail, but the present invention is not limited thereto.
Embodiment 1
Preparation (fermentation liquor treatment) > of < ceramide/glucose ceramide production accelerant
First, prepare in accordance with the following methods substratum: soybean is put into water and soaks 12 hours, make soybean fully absorb moisture, then will absorb the soybean of moisture grind brokenly, by its filtration and remove liquid component, residual residue drains, and obtains bean dregs.These bean dregs are mixed with glucose and water, and make bean dregs and the glucose concentration in mixture reach respectively 10w/v% and 1w/v%, using this mixture as substratum.
Then, the above-mentioned substratum of 100ml is added in Erlenmeyer flask, inoculate a certain amount of abnormal pichia spp (Pichia anomala) (being preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, numbering 360-20-3), at 30 ℃, cultivate 3 days.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample A of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
The evaluation > of < ceramide/glucose ceramide production accelerant
(cell cultures)
First the activation keratinocyte (Cascade Co., Ltd) of, getting normal people is as primary cell, in 75cm
2in culturing bottle (the substratum 2ml that contains somatomedin), 37 ℃, CO
2concentration is culturing cell to 80～90% concentration under 5% condition.Go down to posterity, passage cell is to 175cm
2in square vase, continue culturing cell to 80～90% concentration.Then the human epidermal cell after going down to posterity is inoculated in 12 orifice plates, every pore volume is 2ml, and cell concn is 1 × 10
5individual cell/ml, continues culturing cell to 80～90% concentration.
Then, in above-mentioned 12 orifice plates, change the substratum that does not contain somatomedin, sample A and reference substance (aqueous ethanolic solution that concentration is 50v/v%) are added in above-mentioned 12 orifice plates (n=2), under same culture conditions, cultivate 3 days.
After within 3 days, cultivating, supernatant discarded nutrient solution, with 2ml PBS (phosphate buffered saline buffer), cell being carried out to 2 times cleans, add again the PBS of 1ml to plate hole, to test tube, prepare against follow-up ceramide/glucose ceramide and protein analysis with cell harvestor (cell shovel) collecting cell.
(ceramide/glucose ceramide produces the confirmation of facilitation effect)
The solvent that adds methyl alcohol: chloroform=2.5ml: 1.25ml in the PBS solution that contains cell obtaining in recovery, vibrates 20 minutes, carries out centrifugal (3000rpm, 5 minutes) and separates, thereby obtain supernatant liquor a and precipitation b.Supernatant liquor a, for the quantitative analysis of ceramide/glucose ceramide, is used for precipitation b to the analysis of protein content.
(1) ceramide/glucose ceramide quantitative analysis
The solvent that adds PBS: chloroform=1.25ml: 1.25ml in the supernatant liquor a obtaining thus, vibrates 20 minutes, carries out centrifugal (3000rpm, 5 minutes) and separates, and obtains upper and lower two-phase.Reclaim lower phase, and be dried 40 minutes under 30 ℃, nitrogen protection.In the dry product obtaining, add the chloroform of 100 μ l: the solvent of methyl alcohol=2ml: 1ml dissolves, and it is carried out to the parsing of the amount (be designated as respectively Cer and GlyCer, unit is μ g/ml) of ceramide and glucose ceramide by following thin plate chromatography (TLC) method.
By on ceramide (or glucose ceramide) standard substance and the extremely dry TLC plate of sample solution point, carry out thin plate chromatography take CHCl3: CH3OH: CH3COOH (190: 9: 1v/v/v) as developing agent, treat that developing agent walks to thin plate top, dries up developing agent with blower; Repeat above step once.Then take CHCl3: CH3OH: C3H6O (76: 20: 4v/v/v), as developing agent carries out thin plate chromatography, treats that developing agent walks to apart from 2.5cm place, thin plate bottom, stop exhibition layer, dry up with blower.Spray, with copper sulfate phosphoric acid solution, dries up, and chromatoplate is positioned over and in 180 ℃ of bake plate, toasts colour developing in 7 minutes.Scanning colour developing TLC plate also carries out ceramide (or glucose ceramide) analysis.
(2) analysis of protein content
For the ceramide/glucose ceramide amount in characterize cells, carry out protein content analysis.In above-mentioned precipitation b, add SDS (sodium laurylsulfonate) solution of 90 μ l and mix, at 60 ℃, heat and within 2 hours, make protein denaturation degraded, after cooling, add therein the HCl solution of the 2N of 100 μ l, measure the wherein amount of protein (be designated as Pro, unit is μ g/ml) according to BCA Kit (Protein quantitative analysis test kit) method.
(3) analytical results
For the sample of product of the present invention and reference substance, measure respectively Cer/Pro and GlyCer/Pro (seeing following formula).Take the Cer/Pro of reference substance and GlyCer/Pro as 1, in table 1, represent the relative value of Cer/Pro and the GlyCer/Pro of product of the present invention.In addition, take the amount of the protein in the every hole of reference substance as 100, obtain the relative value (Pro. (%)) of the amount of the protein in every hole of product of the present invention, and obtain accordingly the amount (with respect to the relative quantity of reference substance) of ceramide and the glucose ceramide in every hole,, Cer/Well=Cer/Pro × Pro. (%), GlyCer/Well=GlyCer/Pro × Pro. (%).In table 1, represent in the lump.
If Cer/Pro (or Cer/Well) is greater than 1 (value of reference substance), show that this sample has ceramide and produces facilitation effect; If GlyCer/Pro (or GlyCer/Well) is greater than 1 (value of reference substance), show that this sample has glucose ceramide and produces facilitation effect.
As shown in table 1, confirm that product A of the present invention has ceramide and produces facilitation effect and glucose ceramide generation facilitation effect.
Embodiment 2
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Pichia guilliermondii (Pichia guilliermondii), numbering 422-19-3) replace abnormal pichia spp (Pichia anomala), and after the dehydrated alcohol that adds 2.5 times after fermenting bean dregs in the supernatant liquor obtaining dilutes, mixed solution supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as the sample B of ceramide/glucose ceramide production accelerant of the present invention, C, dry sample is with 50% dissolve with ethanol, by sample B, C is mixed with respectively 1%, the solution of 0.1% concentration, be stored in 4 ℃ of refrigerators as cell cultures additive.In addition, operate similarly to Example 1, and ceramide/glucose ceramide of assess sample B, C generation facilitation effect, result is as shown in table 1.
As shown in table 1, confirm that product B of the present invention, C have ceramide and produce facilitation effect.
Embodiment 3
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Norway's pichia spp (Pichia norvegensis), numbering CICIM Y0151) replace abnormal pichia spp (Pichia anomala), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample D of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as sample E, the F of ceramide/glucose ceramide production accelerant of the present invention, the solution that sample E, F is mixed with respectively to 1%, 0.1% concentration with 50% dissolve with ethanol, is stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to method evaluation sample D similarly to Example 1, ceramide/glucose ceramide of E, F, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample D, E, the F of embodiment 3 has ceramide generation facilitation effect.
Embodiment 4
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with abnormal pichia spp (Pichia anomala), numbering CICIM Y0297) replace abnormal pichia spp (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample G of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as the sample H of ceramide/glucose ceramide production accelerant of the present invention, sample H is mixed with to the solution of 1% concentration with 50% dissolve with ethanol, be stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to ceramide/glucose ceramide of method evaluation sample G, H similarly to Example 1, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample G, the H of embodiment 4 has ceramide and glucose ceramide generation facilitation effect.
Embodiment 5
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with abnormal pichia spp (Pichua anomala), numbering CICIM Y0349) replace abnormal pichia spp (Pichiaanomala) (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample I of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as sample J, the K of ceramide/glucose ceramide production accelerant of the present invention, the solution that sample J, K is mixed with respectively to 1%, 0.1% concentration with 50% dissolve with ethanol, is stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to method evaluation sample I similarly to Example 1, ceramide/glucose ceramide of J, K, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample I, J, the K of embodiment 5 has ceramide and/or glucose ceramide generation facilitation effect.
Embodiment 6
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with film uncut jade pichia spp (Pichia membranifaciens), numbering CICIM Y0360) replace abnormal pichia spp (Pichia anomala) (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample L of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as sample M, the N of ceramide/glucose ceramide production accelerant of the present invention, the solution that sample M, N is mixed with respectively to 1%, 0.1% concentration with 50% dissolve with ethanol, is stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to method evaluation sample L similarly to Example 1, ceramide/glucose ceramide of M, N, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample L, M, the N of embodiment 6 has ceramide generation facilitation effect.
Embodiment 7
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with abnormal pichia spp (Pichia anomala), numbering CICIM Y0420) replace abnormal pichia spp (Pichiaanomala) (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample O of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as sample P, the Q of ceramide/glucose ceramide production accelerant of the present invention, the solution that sample P, Q is mixed with respectively to 1%, 0.1% concentration with 50% dissolve with ethanol, is stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to method evaluation sample O similarly to Example 1, ceramide/glucose ceramide of P, Q, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample O, P, the Q of embodiment 7 has ceramide generation facilitation effect.
Embodiment 8
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with abnormal pichia spp (Pichia anomala), numbering CICIM Y0421) replace abnormal pichia spp (Pichiaanomala) (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample R of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as sample S, the T of ceramide/glucose ceramide production accelerant of the present invention, the solution that sample S, T is mixed with respectively to 1%, 0.1% concentration with 50% dissolve with ethanol, is stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to method evaluation sample R similarly to Example 1, ceramide/glucose ceramide of S, T, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample R, S, the T of embodiment 8 has ceramide generation facilitation effect.
Embodiment 9
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Christian Breton pichia spp (Pichia burtonii), numbering CICIM Y0424) replace abnormal pichia spp (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample U of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as the sample V of ceramide/glucose ceramide production accelerant of the present invention, sample V is mixed with to the solution of 1% concentration with 50% dissolve with ethanol, be stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to ceramide/glucose ceramide of method evaluation sample U, V similarly to Example 1, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample U, the V of embodiment 9 has ceramide generation facilitation effect.
Embodiment 10
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Pichia guilliermondii (Pichia guilliermondii), numbering CICIM Y0440) replace abnormal pichia spp (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample W of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Produce facilitation effect according to ceramide/glucose ceramide of method evaluation sample W similarly to Example 1, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that the sample W of embodiment 10 has glucose ceramide generation facilitation effect.
Embodiment 11
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Pichia guilliermondii (Pichia guilliermondii), numbering CICIM Y0326) replace abnormal pichia spp (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample X of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as the sample Y of ceramide/glucose ceramide production accelerant of the present invention, sample Y is mixed with to the solution of 0.1% concentration with 50% dissolve with ethanol, be stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to ceramide/glucose ceramide of method evaluation sample X, Y similarly to Example 1, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample X, the Y of embodiment 11 has glucose ceramide generation facilitation effect.
Embodiment 12
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Pichia guilliermondii (Pichia guilliermondii), numbering CICIM Y0329) replace abnormal pichia spp (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, gets the sample Z of 1ml mixed solution as ceramide/glucose ceramide production accelerant of the present invention, is stored in 4 ℃ of refrigerators in order to being used as cell cultures additive.
Mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as the sample a of ceramide/glucose ceramide production accelerant of the present invention, sample a is mixed with to the solution of 1% concentration with 50% dissolve with ethanol, be stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to ceramide/glucose ceramide of method evaluation sample Z, a similarly to Example 1, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that sample Z, a of embodiment 12 has ceramide and/or glucose ceramide generation facilitation effect.
Embodiment 13
In the preparation process of ceramide/glucose ceramide production accelerant of embodiment 1, (be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center with Pichia guilliermondii (Pichia guilliermondii), numbering CICIM Y0414) replace abnormal pichia spp (numbering 360-20-3), in addition, cultivate similarly to Example 1.
Then by obtained culture centrifugation (3000rpm, 10 minutes).Residue supernatant liquor adds the dehydrated alcohol dilution of 2.5 times, mixed solution after above-mentioned dehydrated alcohol dilution is carried out to supersound process 10 minutes, centrifugal (3000rpm) 10 minutes, collect supernatant liquor (about 70ml), supernatant liquor obtains filtrate with filter paper filtering, filtrate is dried to constant weight in Vacuumdrier, as the sample b of ceramide/glucose ceramide production accelerant of the present invention, the solution that sample b is mixed with to 1% concentration with 50% dissolve with ethanol, is stored in 4 ℃ of refrigerators as cell cultures additive.
Produce facilitation effect according to ceramide/glucose ceramide of method evaluation sample b similarly to Example 1, result is illustrated in table 1 in the lump.
As shown in table 1, confirm that the sample b of embodiment 13 has ceramide and glucose ceramide generation facilitation effect.
Table 1
In addition, for sample A～Z and a, b, do not joined and above-mentionedly directly carry out the quantitative analysis of ceramide/glucose ceramide containing (that is, not carrying out above-mentioned cell cultures) in 12 orifice plates of keratinocyte, operation is in addition same with above-described embodiment 1 to be carried out.Results verification in sample A～Z and a, b itself, there is no ceramide or glucose ceramide.
Show thus, in product of the present invention itself, there is not ceramide or glucose ceramide, but product of the present invention have ceramide produces facilitation effect and/or glucose ceramide generation facilitation effect, can be used as ceramide and/or glucose ceramide production accelerant and uses.
Claims (12)
1. a manufacture method for ceramide and/or glucose ceramide production accelerant, wherein,
In the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Pichia (Pichia), in clear liquid, obtain from it ceramide production accelerant and/or glucose ceramide production accelerant,
The microorganism of described Pichia (Pichia) is abnormal pichia spp (Pichia anomala), Pichia guilliermondii (Pichia guilliermondii), Norway's pichia spp (Pichia norvegensis), film uncut jade pichia spp (Pichia membranifaciens) or Christian Breton pichia spp (Pichia burtonii).
2. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described carbohydrate is glucose.
3. the manufacture method of ceramide as claimed in claim 2 and/or glucose ceramide production accelerant, wherein,
Described substratum is made up of bean dregs, glucose and water.
4. the manufacture method of ceramide as claimed in claim 3 and/or glucose ceramide production accelerant, wherein,
The content of bean dregs described in described substratum is 8～12w/v%, and the content of described carbohydrate is 0.8～1.2w/v%.
5. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
At 27～35 ℃, carry out described cultivation.
6. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described incubation time is 1～7 day.
7. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
The culture obtaining by described cultivation is carried out centrifugal, thereby obtain described supernatant liquor.
8. the manufacture method of ceramide as claimed in claim 7 and/or glucose ceramide production accelerant, wherein,
In the described supernatant liquor of centrifugal rear acquisition, add after ethanol, set it as ceramide production accelerant and/or glucose ceramide production accelerant.
9. the manufacture method of ceramide as claimed in claim 8 and/or glucose ceramide production accelerant,
Wherein, further to described added the material obtaining after ethanol to refine by ultrasonication, centrifugation, filtration, vacuum drying treatment after, obtain ceramide and/or glucose ceramide production accelerant.
10. the fermented product extract of bean dregs is used for the purposes of the generation that promotes ceramide and/or glucose ceramide,
The fermented product extract of described bean dregs is that the microorganism of cultivating Pichia (Pichia) in the substratum that contains bean dregs and carbohydrate obtains,
The microorganism of described Pichia (Pichia) is abnormal pichia spp (Pichia anomala), Pichia guilliermondii (Pichia guilliermondii), Norway's pichia spp (Pichia norvegensis), film uncut jade pichia spp (Pichia membranifaciens) or Christian Breton pichia spp (Pichia burtonii).
The fermented product extract of 11. bean dregs is as the purposes of ceramide production accelerant and/or glucose ceramide production accelerant,
The fermented product extract of described bean dregs is that the microorganism of cultivating Pichia (Pichia) in the substratum that contains bean dregs and carbohydrate obtains,
The microorganism of described Pichia (Pichia) is abnormal pichia spp (Pichia anomala), Pichia guilliermondii (Pichia guilliermondii), Norway's pichia spp (Pichia norvegensis), film uncut jade pichia spp (Pichia membranifaciens) or Christian Breton pichia spp (Pichia burtonii).
The purposes of the fermented product extract of 12. bean dregs in manufacture ceramide production accelerant and/or glucose ceramide production accelerant,
The fermented product extract of described bean dregs is that the microorganism of cultivating Pichia (Pichia) in the substratum that contains bean dregs and carbohydrate obtains,
The microorganism of described Pichia (Pichia) is abnormal pichia spp (Pichia anomala), Pichia guilliermondii (Pichia guilliermondii), Norway's pichia spp (Pichia norvegensis), film uncut jade pichia spp (Pichia membranifaciens) or Christian Breton pichia spp (Pichia burtonii).
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| CN201110289055.5A CN102465150B (en) | 2010-11-09 | 2011-09-15 | Manufacturing method of ceramide generation accelerator |
| JP2013522070A JP5639713B2 (en) | 2010-11-09 | 2011-11-08 | Method for producing ceramide and / or glucosylceramide production promoter |
| PCT/CN2011/001877 WO2012062043A1 (en) | 2010-11-09 | 2011-11-08 | Method for producing promoter from ceramide and/or glucosylceramide |
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| CN201110289055.5A CN102465150B (en) | 2010-11-09 | 2011-09-15 | Manufacturing method of ceramide generation accelerator |
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