CN102994555A - Manufacturing method of ceramide and/or glucose ceramide production promoter - Google Patents
Manufacturing method of ceramide and/or glucose ceramide production promoter Download PDFInfo
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- CN102994555A CN102994555A CN2011102890080A CN201110289008A CN102994555A CN 102994555 A CN102994555 A CN 102994555A CN 2011102890080 A CN2011102890080 A CN 2011102890080A CN 201110289008 A CN201110289008 A CN 201110289008A CN 102994555 A CN102994555 A CN 102994555A
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Abstract
The invention provides a manufacturing method of a ceramide and/or glucose ceramide production promoter, wherein the ceramide and/or glucose ceramide production promoter is obtained from supernatant fluid of a microorganism of Hansenula, which is cultured in a culture medium containing bean dregs and saccharides. According to the manufacturing method disclosed by the invention, the specific microorganism can be utilized, and a byproduct, namely the bean dregs in the food processing industry and the like can be used as a main raw material of the culture medium, so that the ceramide and/or glucose ceramide production promoter can be obtained with low cost and high efficiency.
Description
Technical field
The present invention relates to utilize specified microorganisms, with the main raw material as substratum such as the by product bean dregs of food-processing industry etc., manufacturing can promote skin to produce ceramide and/or the ceramide of glucose ceramide and/or the method for glucose ceramide production accelerant.
Background technology
The ceramide of one of sphingolipid (Ceramide) is present in the keratoderma, accounts for about 50% of intercellular lipid in the stratum corneum.Sphingolipid (Sphingolipid) is the general name with compound lipid of sphingosine skeleton.Glucose ceramide (Glucosylceramide) is to combine a kind of sphingolipid that glucose forms at ceramide.Temporarily be stored with the form of glucose ceramide or sphingomyelin (Sphingomyelin) by the synthetic ceramide that obtains of epidermic cell; Be discharged to afterwards the extracellular, under the effect of glucose cerebrosidase (Glucocerebrosidase) and sphingomyelinase (Sphingomyelinase), again become ceramide, thereby performance is as the function of intercellular lipid.
All the time, the sphingolipids such as known ceramide, glucose ceramide, galactosyl ceramide have the cuticular maintenance moisture ability of promotion, improve pachylosis texts (patent documentation 1～2).Although skin has the ability that produces ceramide and glucose ceramide, this ability is also insufficient, thereby people have also attempted from the method for external complement ceramide.But the long-term effect of the method can not get approval, and the problem such as existence and stability is low.For example, reported the method (patent documentation 3) of utilizing candida tropicalis (Candida tropicalis) JPCCY0004 strain (NITE P-570) to make the glucose ceramide.But the method is not the method about ceramide and/or glucose ceramide production accelerant, and the method that a large amount of glucose ceramides that exist directly extract in the thalline after only will cultivating; And the ceramide skeleton of the ceramide type that exists in the ceramide skeleton by the synthetic glucose ceramide that obtains of the method and the human body is different, and it has carbon-carbon double bond and have side chain at Liang Chu.Thereby, this glucose ceramide is coated on the human body skin, can not adapt with human body skin and produce the problem of safety in utilization aspect.
And on the other hand, if can strengthen or promote the generation ability of ceramide or the glucose ceramide of skin itself, then need not from external complement ceramide or glucose ceramide, and can improve barrier function and the moisture-keeping functions of skin by promoting skin self generation ceramide or glucose ceramide.Like this, owing to be to produce ceramide or glucose ceramide by skin self, thereby there is not the problem with security aspects such as the adaptability of skin are bad in it.
Therefore, in recent years, quite popular about in keratoderma, studying with the production that promotes the epidermis ceramide to be produced as the material of purpose.As the material that promotes that ceramide produces, that has reported has a ceramide production accelerant (patent documentation 4～6) take the extract of glossy ganoderma, bee pollen (Bee Pollen), Ligusticum wallichii etc. as effective constituent.Yet, because these ceramide production accelerants all are to extract from plant material to obtain, this extractions operation length that expends time in, and utilize the reasons such as possibility is limited, its manufacturing cost height owing to natural resource.
In addition, also reported ceramide synthesis accelerators (patent documentation 7) take the lactic acid bacteria culture that contains nicotinic acid and/or niacinamide or Yeast culture as effective constituent.Yet, can find out from the embodiment of patent documentation 7, do not contain the lactic acid bacteria culture of nicotinic acid and/or niacinamide or the ceramide generation of Yeast culture (comparative example 1～4) and there is no considerable change than control group, thereby this ceramide synthesis accelerators contains in fact nicotinic acid and/or niacinamide as effective constituent, the synthetic facilitation effect of its ceramide is abundant not enough, the leeway that still is improved.And, owing to use nicotinic acid or niacinamide, thereby its manufacturing cost is higher.
In addition, the large multipotency of the microorganism of known Hansenula (Hansenula) produces ethyl acetate, thereby increases product fragrance, therefore is generally used for wine brewing and foodstuffs industry.
But, also do not utilize at present the microorganism of Hansenula (Hansenula) to make the report of ceramide and/or glucose ceramide production accelerant.
Patent documentation 1: Japanese kokai publication sho 61-260008 communique
Patent documentation 2: Japanese kokai publication sho 61-271205 communique
Patent documentation 3: TOHKEMY 2005-194240 communique
Patent documentation 4: TOHKEMY 2010-70499 communique
Patent documentation 5: TOHKEMY 2010-150237 communique
Patent documentation 6: Japanese kokai publication hei 9-194383 communique
Patent documentation 7: TOHKEMY 2010-22217 communique
Summary of the invention
For the technical problem that exists in the above-mentioned prior art, the inventor has carried out concentrated research, found that: the microorganism of in the substratum that contains bean dregs and carbohydrate, cultivating Hansenula (Hansenula), particularly cultivate not debaryomyces hansenii (Hansenula ciferrii) of multiple-shaped nuohan inferior yeast (Hansenula polymorpha) or west, can be low-cost and obtain efficiently ceramide and/or glucose ceramide production accelerant, thus the present invention finished.
The present invention relates to, the manufacture method of a kind of ceramide and/or glucose ceramide production accelerant is provided, wherein, in the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Hansenula (Hansenula), obtain from it ceramide and/or glucose ceramide production accelerant in the clear liquid.
The invention still further relates to, the purposes of the non-therapeutic purpose of the generation that the fermented product extract of the Hansenula (Hansenula) of bean dregs be used for to promote ceramide and/or glucose ceramide is provided.
The invention still further relates to, provide the fermented product extract of Hansenula (Hansenula) of bean dregs as the purposes of the non-therapeutic purpose of ceramide and/or glucose ceramide production accelerant.
The invention still further relates to, the purposes of fermented product extract in making ceramide and/or glucose ceramide production accelerant of the Hansenula (Hansenula) of bean dregs is provided.
The invention effect
Manufacturing method according to the invention, can utilize specified microorganisms, with the main raw material as substratum such as the by product bean dregs of food-processing industry etc., low-cost and obtain efficiently ceramide and/or glucose ceramide production accelerant, the generation ceramide that this promotor can Effective Raise skin has and/or the ability of glucose ceramide, thereby the moisture-retaining capacity of Effective Raise skin.
Embodiment
(can be used for the microorganism that ceramide and/or glucose ceramide production accelerant are made)
As the microorganism of the Hansenula (Hansenula) that uses in the present invention, can list at least a kind of microorganism that the multiple-shaped nuohan inferior yeast (Hansenula polymorpha) that uses from field of food, west are not selected debaryomyces hansenii (Hansenula ciferrii), inferior film debaryomyces hansenii (Hansenula subpelliculosa), Saturn debaryomyces hansenii (Hansenula saturnus), the Hansenula anomala (Hansenula anomala) etc.; The variant that also comprises these microorganisms that obtain according to usual method.These microorganisms are as described below to play a role, and by cultivating in the substratum that contains bean dregs and carbohydrate, thereby can be improved from it the ceramide of epidermic cell and/or the material that the glucose ceramide produces ability in the clear liquid.Wherein, has the viewpoint that higher ceramide produces facilitation effect and glucose ceramide generation facilitation effect from the product that obtains, preferred multiple-shaped nuohan inferior yeast (Hansenula polymorpha) or west be debaryomyces hansenii (Hansenula ciferrii) not, wherein, the synonym/former name of multiple-shaped nuohan inferior yeast (Hansenula polymorpha) has: Angus pichia spp (Pichia angusta), Angus debaryomyces hansenii (Hansenula angusta), multiform Europe adds iron bacteria (Ogataea polymorpha).
The microorganism of Hansenula involved in the present invention (Hansenula) can be bought from commercial channel such as Chinese common micro-organisms preservation administrative center (CGMCC), Chinese Typical Representative culture collection center (CCTCC), Southern Yangtze University's industrial microorganism resource and information centers (CICIM) and obtain.
(manufacture method of ceramide and/or glucose ceramide production accelerant)
The manufacture method of ceramide of the present invention and/or glucose ceramide production accelerant is cultivated mentioned microorganism by in the substratum that contains bean dregs and carbohydrate, and obtains ceramide and/or glucose ceramide production accelerant in the clear liquid from it.The concrete steps of the method are as follows.
＜substratum 〉
In the present invention, cultivate one kind or two or more in the mentioned microorganism in the preferred liquid medium within.
In the present invention, the main component of liquid nutrient medium is bean dregs and carbohydrate.Also can suitably add peptone, yeast powder.
Because bean dregs and carbohydrate all are the materials that obtains easily, thereby substratum of the present invention has the low advantage of cost.
Wherein, bean dregs refer to, in water, make it fully absorb moisture soybeans soaking after, again through pulverizing, removing by filter water and the material that obtains; As mentioned above, especially can use the bean dregs that are left after the squeezing soya-bean milk in the food industries.
As carbohydrate, can use separately in glucose, maltose, the sucrose etc. any one, perhaps mix and use.From improving the viewpoint of fermentative production and usability, preferred glucose wherein.
In the present invention, ceramide produces facilitation effect and/or the glucose ceramide produces facilitation effect in order to improve, medium optimization of the present invention is made of bean dregs, glucose and water, further the content of the bean dregs in the preferred culture medium is 5～20w/v%, 8～12w/v% more preferably, and the content of glucose is 0.2～2w/v%, 0.8～1.2w/v% more preferably.
Below, above-mentioned this substratum for the manufacture of ceramide and/or glucose ceramide production accelerant is called " ceramide/glucose ceramide production accelerant is made substratum ".
＜culture condition 〉
The thalline direct inoculation of mentioned microorganism is made in the substratum to ceramide/glucose ceramide production accelerant.As the culture condition for the manufacture of ceramide and/or glucose ceramide production accelerant, from the necessary temperature of growth, the time of optimization Hansenula (Hansenula) microorganism, and the viewpoint that improves ceramide generation facilitation effect and glucose ceramide generation facilitation effect is set out, preferably 20～40 ℃, more preferably under 27～35 ℃ culture temperature, cultivated 1～7 day, the preferred cultivation 48～96 hours more preferably cultivated 68～76 hours.
The recovery of＜ceramide and/or glucose ceramide production accelerant, refining 〉
From the nutrient solution that above-mentioned cultivation obtains, reclaim, make with extra care ceramide and/or glucose ceramide production accelerant, can as required, suitably make up the common operations such as filtration, centrifugation, ion-exchange or adsorption chromatography, solvent extraction, crystallization.Particularly, for example, can from nutrient solution, remove thalline by centrifugation, in ultrasonic treatment unit, pulverize again, reclaim the supernatant liquor after centrifugal.Then, the supernatant liquid filtering after will reclaiming is again removed impurity etc., obtains the fermenting bean dregs extract under the vacuum after the drying treatment as ceramide of the present invention and/or glucose ceramide production accelerant.
Shown in following embodiment, by cultivating Hansenula of the present invention (Hansenula) microorganism, and the fermenting bean dregs extract that from culture supernatant, obtains, in normal human's keratinocyte, have the effect that the amount that makes ceramide and/or glucose ceramide increases.
Therefore, by the fermenting bean dregs extract of cultivating Hansenula of the present invention (Hansenula) microorganism and from culture supernatant, obtaining, can be used for promoting ceramide and/or the therapeutic purpose of glucose ceramide generation or the use of non-therapeutic purpose, the ceramide and/or the glucose ceramide production accelerant that also can be used as therapeutic purpose or non-therapeutic purpose use.Using of non-therapeutic purpose is the use as purpose of improving looks, or for the use of keeping state of health etc.And the use of described therapeutic purpose and the use of non-therapeutic purpose can fully be carried out with distinguishing.
In addition, by the fermenting bean dregs extract of cultivating Hansenula of the present invention (Hansenula) microorganism and from culture supernatant, obtaining, can also be for the manufacture of ceramide and/or glucose ceramide production accelerant.
Ceramide of the present invention and/or glucose ceramide production accelerant can be used as the ceramide that makes in the stratum corneum and/or the glucose ceramide increases, and recovers or keep the uses such as the barrier function of skin and the pharmaceuticals of moisture-keeping functions, accurate pharmaceuticals, makeup.In addition, ceramide production accelerant of the present invention or glucose ceramide production accelerant also can be used as take ceramide produce to promote or the glucose ceramide produce promote as concept, and indicate that as required the accurate pharmaceuticals of this concept, makeup use.
Use form as ceramide of the present invention and/or glucose ceramide production accelerant, can be used as the additive that adds in the culturing cell, perhaps make the external composition for skin such as detergent, makeup, can provide with forms such as astringent, emulsion, gel, frost, ointment, powder, particles according to using method.When the various external composition for skin of preparation, can use individually ceramide of the present invention and/or glucose ceramide production accelerant, or appropriate combination usually uses such as the oiliness composition of cooperation, 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 in the external composition for skin such as detergent and makeup.
In the epidermic cell that ceramide of the present invention and/or glucose ceramide production accelerant is added to cultivation, promote in the situation of ceramide and/or glucose ceramide, as fermenting bean dregs extract of the present invention (being scaled the drying solid composition), its addition is preferably 0.0001～10w/v%, more preferably 0.001～5w/v%.When addition during greater than 0.0001w/v%, can obtain sufficient facilitation effect; And when addition during less than 10w/v%, less to the pungency of epidermic cell.
When ceramide of the present invention and/or glucose ceramide production accelerant being engaged in external composition for skin and using, thereby from the moisture-retaining capacity of the generation Effective Raise skin of effective promotion ceramide and/or glucose ceramide and be difficult for demonstrating the color of culture and the viewpoint of smell, drying solid composition as the fermenting bean dregs extract, its use level is preferably 0.01～20w/v% of external composition for skin total amount, more preferably 0.1～10w/v%.
Embodiment
Below, the present invention will be described in more detail based on embodiment, but the present invention is not limited thereto.
Embodiment 1
(manufacturing of ceramide and/or glucose ceramide production accelerant)
As the microorganism of making ceramide and/or glucose ceramide production accelerant from bean dregs, use multiple-shaped nuohan inferior yeast (Hansenula polymorpha) (to buy from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIM Y0234).
The above-mentioned bacterial strains that will obtain after will cultivating under 30 ℃ in YPD (Yeast Extract Peptone Dextrose Medium) substratum is inoculated in the Erlenmeyer flask (capacity 300ml) that contains ceramide/glucose ceramide production accelerant manufacturing substratum 100ml, 30 ℃ of lower cultivations 72 hours.This substratum contains 10w/v% bean dregs and 1w/v% glucose.Wherein, the preparation of bean dregs was soaked 12 hours by soybean being put into water, made soybean fully absorb moisture, and the soybean that then will absorb moisture is ground broken, after filtering and removing liquid component, residual residue was drained to obtain.
Then, in order from the nutrient solution that obtains, to reclaim ceramide and/or glucose ceramide production accelerant, removed thalline in 10 minutes with the 3000rpm centrifugation, supernatant liquor after the recovery centrifugation, the dehydrated alcohol that adds 2.5 times in the culture supernatant after recovery, use again ultrasonic treatment unit (manufacturing of Branson company) to pulverize 10 minutes, with 3000rpm centrifugation 30 minutes, reclaim supernatant liquor.With the about 70ml of this supernatant liquor of filter paper filtering, dry this filtered liquid obtains the fermenting bean dregs extract, as ceramide of the present invention and/or glucose ceramide production accelerant to constant weight in Vacuumdrier below 40 ℃ again.
And then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjustment fermenting bean dregs extract concentrations is 0.1w/v% (the present invention's product A-1), being kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes to test.
Embodiment 2
As the microorganism of making ceramide/glucose ceramide production accelerant from bean dregs, use multiple-shaped nuohan inferior yeast (Hansenula polymorpha) (to buy from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIM Y0205).Adopt the method identical with embodiment 1 to obtain the fermenting bean dregs extract, behind ceramide of the present invention and/or glucose ceramide production accelerant (the present invention's product B-1), be kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes test.
Embodiment 3
As the microorganism of making ceramide/glucose ceramide production accelerant from bean dregs, use multiple-shaped nuohan inferior yeast (Hansenula polymorpha) (to buy from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0257).Adopt the method identical with embodiment 1 to obtain the fermenting bean dregs extract, as ceramide of the present invention and/or glucose ceramide production accelerant, and then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjusting its concentration and being 0.1w/v% (the present invention's product C-1), be kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes test.
Embodiment 4
As the microorganism of making ceramide/glucose ceramide production accelerant from bean dregs, use multiple-shaped nuohan inferior yeast (Hansenula polymorpha) (to buy from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0122).Adopt the method identical with embodiment 1 to obtain the fermenting bean dregs extract, as ceramide of the present invention and/or glucose ceramide production accelerant (the present invention's product D-1), and then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjusting respectively its concentration and being 1w/v% (the present invention's product D-2), 0.1w/v% (the present invention's product D-3), be kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes test.
Embodiment 5
As the microorganism of making ceramide/glucose ceramide production accelerant from bean dregs, use multiple-shaped nuohan inferior yeast (Hansenula polymorpha) (to buy from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0135).Adopt the method identical with embodiment 1 to obtain the fermenting bean dregs extract, behind ceramide of the present invention and/or glucose ceramide production accelerant (the present invention's product E-1), be kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes test.
Embodiment 6
As the microorganism of making ceramide/glucose ceramide production accelerant from bean dregs, use west not debaryomyces hansenii (Hansenula ciferrii) (is bought from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIM Y0150).Adopt the method identical with embodiment 1 to obtain the fermenting bean dregs extract, as ceramide of the present invention and/or glucose ceramide production accelerant, and then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjusting its concentration and being 1w/v% (the present invention's product F-1), be kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes test.
Embodiment 7
As the microorganism of making ceramide/glucose ceramide production accelerant from bean dregs, use multiple-shaped nuohan inferior yeast (Hansenula polymorpha) (to buy from Southern Yangtze University, and be preserved in Chinese Universities ' industrial microorganism resource and the CICIM of information center, deposit number CICIMY0197).Adopt the method identical with embodiment 1 to obtain the fermenting bean dregs extract, as ceramide of the present invention and/or glucose ceramide production accelerant (the present invention's product G-1), and then, with this fermenting bean dregs extract of 50v/v% dissolve with ethanol, after adjusting respectively its concentration and being 1w/v% (the present invention's product G-2), 0.1w/v% (the present invention's product G-3), be kept under 4 ℃, the ceramide after being used for/glucose ceramide produces and promotes test.
(ceramide/glucose ceramide produces and promotes test)
＜keratinocyte (Keratinocytes) is cultivated 〉
At first, activation keratinocyte (Cascade Co., Ltd) is as primary cell, in 75cm
2In the culturing bottle (the substratum 2ml that contains somatomedin), at 37 ℃, CO
2Concentration is to carry out cell cultures under 5% the condition, is 80～90% until cultivate to cell aggregation density (confluence).Passage cell is to 175cm again
2Continue culturing cell to 80～90% concentration in the square vase.Then with 1 * 10
5Individual cell/ml is passaged in 12 orifice plates (every pore volume is 2ml), continues culturing cell to 80～90% concentration.
Then, change the substratum that does not contain somatomedin, (n=2) adds respectively the aqueous ethanolic solution of the fermenting bean dregs extract that obtains in above-described embodiment 1～7 with the amount in 20 μ l/ holes in above-mentioned 12 orifice plates.In addition, only add in contrast product of 50v/v% ethanol (not adding ceramide and/or glucose ceramide production accelerant), cultivated 72 hours under the same culture conditions.Cultivate after 72 hours, the supernatant discarded nutrient solution, clean twice in cell (2ml/ hole) with 2ml PBS (phosphate buffered saline buffer), add again 1ml PBS to plate hole, to test tube, prepare against follow-up ceramide/glucose ceramide and protein analysis with cell harvestor (cell shovel) collecting cell.
＜lipids extraction 〉
Lipids extraction adopts Bligh﹠amp; The Dyer method is carried out.Particularly, at first, in the celliferous PBS liquid that above-mentioned recovery obtains, add the solvent of methyl alcohol: chloroform=2.5ml: 1.25ml, vibrated 20 minutes, carry out centrifugation (3000rpm, 5 minutes), thereby obtain supernatant liquor and precipitation.Supernatant A is transferred in the other test tube for ceramide/glucose ceramide quantitative analysis, with the analysis of deposit B for protein content.
＜protein content analysis 〉
For the ceramide in the characterize cells/glucose ceramide amount, carry out the protein content analysis.In the deposit B of above-mentioned recovery, add SDS (sodium lauryl sulphate) solution of 900 μ l and mix, heating made the protein denaturation degraded in 2 hours under 60 ℃ of water-baths, add therein the 2N HCl solution of 100 μ l after the cooling, measure the wherein amount of protein (be designated as Pro, unit is μ g/ml) according to BCAKit (BCA determination of protein concentration test kit) method.
＜ceramide/glucose ceramide component analysis 〉
The solvent that in residual supernatant A, adds PBS: chloroform=1.25ml: 1.25ml; vibrate 20 minutes; 3000rpm carried out centrifugation 5 minutes; separate, reclaim the chloroform layer as lower phase; and dry chloroform layer obtained dry product in 40 minutes under 30 ℃, nitrogen protection; the solvent of the chloroform that adds 100 μ l: methyl alcohol=2.5ml: 1.25ml; and with following thin plate chromatography (TLC) method the amount (be designated as respectively Cer and GlyCer, unit is μ g/ml) of ceramide and glucose ceramide is carried out quantitatively.
Adopt dry TLC plate, take chloroform: methyl alcohol: acetic acid (190: 9: 1v/v/v) carry out the thin plate chromatography as developing agent, treat that developing agent walks to the thin plate top, dries up developing agent with blower; Repeat above step once.Then take chloroform: methyl alcohol: acetone (76: 20: 4v/v/v) carry out the thin plate chromatography as developing agent, treat that developing agent walks to apart from 2.5cm place, thin plate bottom, stop the exhibition layer, dry up with blower.Spray dries up with the copper sulfate phosphoric acid solution, and chromatoplate is positioned over baking colour developing in 7 minutes on 180 ℃ of bake plate.Scanning colour developing TLC plate also carries out ceramide (or glucose ceramide) analysis.
Then, according to following formula (1), income value respectively divided by separately protein content, is obtained Cer/Pro and GlyCer/Pro.Take the Cer/Pro of reference substance and GlyCer/Pro as 1, obtain the relative value of Cer/Pro of the present invention and GlyCer/Pro.And the amount of the protein in the every hole of reference substance is as 100, obtains ceramide in every hole and the amount (being designated as respectively Cer/Well and GlyCer/Well, with respect to the relative quantity of reference substance) of glucose ceramide.In the lump expression in table 1.
Formula (1)
Cer/Pro=ceramide amount (μ g/ml)/protein content (μ g/ml)
GlyCer/Pro=glucose ceramide amount (μ g/ml)/protein content (μ g/ml)
Table 1
As shown in table 1, the present invention's product that obtain among the embodiment 2 had both had ceramide and had produced facilitation effect, had again the glucose ceramide and produced facilitation effect; In addition, it is remarkable that the ceramide of the present invention's product that obtain among embodiment 1, embodiment 3 and the embodiment 6 produces facilitation effect, and the glucose ceramide of the present invention's product that obtain among embodiment 4 and the embodiment 7 generation facilitation effect is remarkable.Therefore, confirmed that ceramide of the present invention and/or glucose ceramide production accelerant have ceramide and produce facilitation effect and/or glucose ceramide generation facilitation effect.
(not having the test of ceramide and glucose ceramide in ceramide of the present invention and/or the glucose ceramide production accelerant)
Without cell cultures, directly adopt the TLC method that the ceramide and/or the glucose ceramide production accelerant that obtain in above-described embodiment 1～7 are analyzed, the result shows does not have ceramide and glucose ceramide to exist in the present invention's product.
Show thus, although do not have ceramide or glucose ceramide in the product of the present invention itself, ceramide produces facilitation effect and/or the glucose ceramide produces facilitation effect but have, and can be used as ceramide and/or glucose ceramide production accelerant and uses.
Claims (13)
1. the manufacture method of a ceramide and/or glucose ceramide production accelerant, wherein,
In the substratum that contains bean dregs and carbohydrate, cultivate the microorganism of Hansenula (Hansenula), obtain from it ceramide and/or glucose ceramide production accelerant in the clear liquid.
2. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
The microorganism of described Hansenula (Hansenula) is not debaryomyces hansenii (Hansenula ciferrii) of multiple-shaped nuohan inferior yeast (Hansenula polymorpha) or west.
3. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described carbohydrate is glucose.
4. the manufacture method of ceramide as claimed in claim 3 and/or glucose ceramide production accelerant, wherein,
Described substratum is made of bean dregs, glucose and water.
5. the manufacture method of ceramide as claimed in claim 4 and/or glucose ceramide production accelerant, wherein,
The content of bean dregs described in the described substratum is 8～12w/v%, and the content of described carbohydrate is 0.8～1.2w/v%.
6. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described culture temperature is 27～35 ℃.
7. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
Described incubation time is 1～7 day.
8. the manufacture method of ceramide as claimed in claim 1 and/or glucose ceramide production accelerant, wherein,
To be undertaken by the nutrient solution that described cultivation obtains centrifugal, thereby obtain described supernatant liquor.
9. the manufacture method of ceramide as claimed in claim 8 and/or glucose ceramide production accelerant, wherein,
After in the described supernatant liquor of centrifugal rear acquisition, adding ethanol, further after ultrasonication, centrifugation, filtration, vacuum drying treatment are made with extra care, obtain ceramide and/or glucose ceramide production accelerant.
10. the fermenting bean dregs extract be used for to promote the purposes of non-therapeutic purpose of the generation of ceramide and/or glucose ceramide,
Described fermenting bean dregs extract is that the microorganism of cultivating Hansenula (Hansenula) in containing the substratum of bean dregs obtains.
11. the fermenting bean dregs extract is as the purposes of the non-therapeutic purpose of ceramide and/or glucose ceramide production accelerant,
Described fermenting bean dregs extract is that the microorganism of cultivating Hansenula (Hansenula) in containing the substratum of bean dregs obtains.
12. the purposes of fermenting bean dregs extract in making ceramide and/or glucose ceramide production accelerant,
Described fermenting bean dregs extract is that the microorganism of cultivating Hansenula (Hansenula) in containing the substratum of bean dregs obtains.
13. such as the described purposes of any one in the claim 10～12, wherein,
The microorganism of described Hansenula (Hansenula) is not debaryomyces hansenii (Hansenula ciferrii) of multiple-shaped nuohan inferior yeast (Hansenula polymorpha) or west.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110289008.0A CN102994555B (en) | 2011-09-15 | 2011-09-15 | Manufacturing method of ceramide and/or glucose ceramide production promoter |
| 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110289008.0A CN102994555B (en) | 2011-09-15 | 2011-09-15 | Manufacturing method of ceramide and/or glucose ceramide production promoter |
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| CN102994555B CN102994555B (en) | 2014-10-08 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11322534A (en) * | 1998-05-14 | 1999-11-24 | Kanebo Ltd | Ceramide synthesis accelerator |
| CN1309718A (en) * | 1998-07-03 | 2001-08-22 | 科斯莫弗姆有限公司 | Improved microbial strains producing sphingoid bases |
| CN101653537A (en) * | 2008-08-18 | 2010-02-24 | 复旦大学附属中山医院 | Ceramide production accelerant |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11322534A (en) * | 1998-05-14 | 1999-11-24 | Kanebo Ltd | Ceramide synthesis accelerator |
| CN1309718A (en) * | 1998-07-03 | 2001-08-22 | 科斯莫弗姆有限公司 | Improved microbial strains producing sphingoid bases |
| CN101653537A (en) * | 2008-08-18 | 2010-02-24 | 复旦大学附属中山医院 | Ceramide production accelerant |
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| CN102994555B (en) | 2014-10-08 |
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