CN104232724A - Method of preparing yellow ginger diosgenin by microbial conversion method - Google Patents
Method of preparing yellow ginger diosgenin by microbial conversion method Download PDFInfo
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- CN104232724A CN104232724A CN201410330905.5A CN201410330905A CN104232724A CN 104232724 A CN104232724 A CN 104232724A CN 201410330905 A CN201410330905 A CN 201410330905A CN 104232724 A CN104232724 A CN 104232724A
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Abstract
The invention provides a method of preparing yellow ginger diosgenin by a microbial conversion method. The method specifically comprises the following steps: extracting yellow ginger saponin by 75% alcohol; adding the yellow ginger saponin as well as a nitrogen source and other inorganic salt ions to prepare a seed activation culture medium and a fermentation culture medium; inoculating aspergillus awamori and fermenting a shaking bed; and adding methanol to ultrasonically oscillate and extract diosgenin produced by fermentation. The method disclosed by the invention is high conversation ratio of yellow ginger diosgenin, environmentally friendly and capable of recycling the methanol; and moreover, the defects that the existing acid hydrolysis process wastes raw materials, is severe in pollution and high in investment cost are solved.
Description
Technical field
The present invention relates to the preparation method of yellow ginger diosgenin, be specifically related to a kind of method that microbe transformation method prepares yellow ginger diosgenin.
Background technology
Diosgenin is the important foundation raw material producing steroidal drug.The diosgenin produced through intestinal flora metabolism after dioscin is oral is the real effective constituent that plays a role and is the important medicine chemical material of synthesizing steroid hormone medicine and contraceptive steroid, also has anti-ageing, antitumor, Killing Oncomelania Hupensis, prevention and cure of cardiovascular disease effect simultaneously.
The main technique of producing diosgenin at present both at home and abroad has direct acid-hydrolysis method and pre fermentation acid-hydrolysis method.These two kinds of techniques all have this operation of acid hydrolysis, and namely acid-hydrolysis method has its hydrolysis time short, the advantage that cost is low, but shortcoming is obvious equally, acid-hydrolysis method can produce the sewage of high OD value, and hydrolytic process has part saponin to be destroyed, and this does not all meet the Scientific Outlook on Development of environmental protection.
Recent study person has been found that aspergillus oryzae, the saponin(e in yellow ginger can be converted into diosgenin by wood some microorganisms such as mould, but its fermentation conversion rate all can not reach desirable requirement, reason is that the substrate in fermention medium is mostly the yellow ginger saponin(e of yellow ginger powder or dehydrated alcohol extraction, starch on the one hand in yellow ginger powder and cellulosic content have accounted for more than 80%, and this just hinders the hydrolytic action of microorganism to saponin(e; On the other hand, dehydrated alcohol can only the saponin(e of Extraction parts alcohol dissolubility, and water miscible saponin(e can lose.Therefore the aqueous ethanol that the present invention utilizes response surface optimization method to obtain can extract the saponin(e in yellow ginger efficiently, this microbe conversion effect being microorganism is provided convenience, most of yellow ginger saponin(e that alcohol extracting can obtain by the strain Aspergillus awamori simultaneously utilizing screening to obtain is converted into diosgenin, is conducive to further suitability for industrialized production to its research.
Summary of the invention
A kind of microbe transformation method is the object of the present invention is to provide to prepare the method for yellow ginger diosgenin.
The Classification And Nomenclature of Aspergillus awamori used in the present invention is that Aspergillus awamori (Aspergillus awamori) black tangerine is antibacterial, by China typical culture collection center preservation, address is: Wuhan, China Wuhan University, deposit number is CCTCC NO:M2013639, and preservation date is on December 8th, 2013.
Object of the present invention is achieved through the following technical solutions:
Microbe transformation method prepares a method for yellow ginger diosgenin,
(1) preparation of carbon source yellow ginger saponin(e, in seed activation substratum and fermention medium, carbon source is yellow ginger saponin(e, obtains the optimised process of the extraction of yellow ginger saponin(e with response phase method optimization;
(2) Aspergillus awamori is seeded in seed activation substratum carries out microbe conversion;
(3) ferment in shaking table mode;
(4) separating-purifying obtains yellow ginger diosgenin.
Microbe transformation method prepares a method for yellow ginger diosgenin, comprises the steps:
(1) yellow ginger is washed with water totally in 45 DEG C ~ 50 DEG C oven for drying to constant weight, then pulverize 300 ~ 400 mesh sieves with powder beater and obtain yellow ginger raw material powder, add the extraction of ethanolic soln shaker water bath and obtain carbon source yellow ginger saponin(e for 2 ~ 3 times, add in seed activation substratum and fermention medium;
(2) Aspergillus awamori is seeded in seed activation substratum cultivates, obtain the Aspergillus awamori activated;
(3) be that 1% ~ 2% stroke-physiological saline solution dissolving is prepared into bacteria suspension by the Aspergillus awamori mass percent concentration that step (2) activates, be then seeded in fermention medium, ferment in shaking table mode, obtain yellow ginger saponin(e fermented liquid;
(4) fermentation liquor centrifuging and taking precipitation, adds organic solvent and utilizes sonic oscillation to extract, obtain yellow ginger diosgenin.
In aforesaid method, described in step (1), the solid-liquid ratio of yellow ginger raw material powder and ethanolic soln is 1:4 ~ 1:7g/ml; The concentration of volume percent of described ethanolic soln is 70% ~ 80%; Described shaker water bath Extracting temperature is 70 DEG C ~ 80 DEG C, and rotating speed is 180 ~ 200r/min, and this extracting method, through response phase method optimization, extracts yield high.
In aforesaid method, the described seed activation substratum composition of step (2): carbon source yellow ginger saponin(e 2-4g/L, peptone 6-8g/L or SODIUMNITRATE 3-5g/L, potassium primary phosphate 1-3g/L, 7H
2o magnesium sulfate 0.5-1.5g/L, Repone K 0.5-1.5g/L, 7H
2o ferric sulfate 0.01-0.03g/L, agar powder 18-22g/L, pH6.5-7.5; Described cultivated days is 5 ~ 7 days.It is first Application that the Aspergillus awamori that the present invention uses is prepared in yellow ginger diosgenin at microbe transformation method, has the feature of high conversion.
In aforesaid method, the fermentation condition described in step (3) is the fermention medium of the bottled 80-100ml of taper, and inoculum size is 5%-8%, culture temperature 28-35 DEG C, shaking speed 180-200r/min by volume, cultivates 5-7 days; Described fermention medium composition: carbon source yellow ginger saponin(e 2-4g/L, peptone 6-8g/L or SODIUMNITRATE 3-5g/L, potassium primary phosphate 1-3g/L, bitter salt 0.5-1.5g/L, Repone K 0.5-1.5g/L, seven ferric sulfate hydrate 0.01-0.03g/L, pH6.5-7.5, sterilizing 15min under 121 DEG C of high pressure steam.
In aforesaid method, the described concrete grammar utilizing sonic oscillation to carry out extracting of step (4) is for adding organic solvent, and extract in the mode of sonic oscillation, ultrasonic power is 200 ~ 320w, vibration rotating speed 180r/min-200r/min, extraction time 2-3h; Described organic solvent comprises anhydrous methanol or dehydrated alcohol, and the volume ratio of organic solvent and yellow ginger saponin(e fermented liquid is 2:1-3:1.
The present invention has the following advantages and effect relative to prior art tool:
1. Aspergillus awamori has higher transformation efficiency.
2. first utilize ethanol to be extracted efficiently by the saponin(e in yellow ginger to ferment again, decrease Mierocrystalline cellulose in raw material and starch to the package action of saponin(e, improve the fermentation efficiency of Aspergillus awamori, strengthen the specific aim of fermenting substrate; Tunning utilizes methyl alcohol to carry out supersound extract, and the common sherwood oil water-bath refluxing extraction of efficiency ratio improves greatly, and environmental friendliness, the recyclable recycling of methyl alcohol; Overcome existing acid hydrolysis process to waste raw material, seriously polluted, the shortcoming that cost of investment is high.
3. with existing with compared with yellow ginger powder or the dehydrated alcohol extraction yellow ginger saponin(e fermentation mode that is substrate, the saponin(e that can make full use of in yellow ginger of the present invention, improves the specificity of fermentation substrate.
Accompanying drawing explanation
Fig. 1 is solvent HPLC collection of illustrative plates in HPLC;
Fig. 2 is diosgenin reference substance HPLC collection of illustrative plates;
Fig. 3 is Aspergillus awamori converted product HPLC collection of illustrative plates;
Fig. 4 is alcohol extract acid hydrolysis products HPLC collection of illustrative plates;
Fig. 5 is diosgenin reference substance typical curve.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the invention mode is not limited thereto.
The Aspergillus awamori used in embodiment is by China typical culture collection center preservation, and deposit number is CCTCC NO:M2013639, and preservation date is on December 8th, 2013.
The preparation method of the yellow ginger raw material powder in embodiment is: washed with water by yellow ginger clean in 45 DEG C of oven for drying to constant weight, then pulverized 400 mesh sieves with powder beater and obtain yellow ginger raw material powder.
Embodiment 1
Microbe transformation method prepares a method for yellow ginger diosgenin, comprises the following steps:
(1) in seed activation substratum and fermention medium, carbon source is yellow ginger saponin(e, with response phase method optimization obtain yellow ginger saponin(e the optimised process of extraction.Take ethanol 200r/min shaker water bath at 70 DEG C that 10g yellow ginger powder adds 75% (v/v) according to the solid-liquid ratio of 1:5 (g/ml) and extract three yellow ginger powder, obtain carbon source yellow ginger saponin(e, be applied in seed activation substratum and fermention medium.
(2) inoculate Aspergillus awamori and carry out microbe conversion.Be inoculated in seed activation culture medium culturing 5 days by screening the Aspergillus awamori obtained, seed activation substratum forms: carbon source yellow ginger saponin(e 2g/L, peptone 6g/L, potassium primary phosphate 1/L, MgSO
47H
2o0.5g/L, Repone K 0.5g/L, Fe
2(SO4)
37H
2o0.01g/L, agar powder 18g/L, pH7.0, sterilizing 15min under 121 DEG C of high pressure steam.
(3) ferment in shaking table mode.Fermentation condition is the fermention medium of the bottled 100ml of 500ml taper, and inoculum size is 5% by volume, culture temperature 30 DEG C, shaking speed 180r/min, cultivates 7 days.Fermention medium forms: yellow ginger saponin(e 2g/L, peptone 6g/L, potassium primary phosphate 1/L, bitter salt 0.5g/L, Repone K 0.5g/L, seven ferric sulfate hydrate 0.01g/L, pH7.0, sterilizing 15min under 121 DEG C of high pressure steam.
(4) fermentation liquor centrifuging and taking precipitation, adds organic solvent and utilizes sonic oscillation to carry out extraction diosgenin.Be that 1:2 ratio adds anhydrous methanol in yellow ginger saponin(e fermented liquid and anhydrous methanol volume ratio, extract in the mode of sonic oscillation, ultrasonic power is 100W, and vibration rotating speed 180r/min, extraction time 3h, obtains yellow ginger diosgenin.
Embodiment 2
Microbe transformation method prepares a method for yellow ginger diosgenin, comprises the following steps:
(1) in seed activation substratum and fermention medium, carbon source is yellow ginger saponin(e, with response phase method optimization obtain yellow ginger saponin(e the optimised process of extraction.Take ethanol 200r/min shaker water bath at 75 DEG C that 10g yellow ginger powder adds 80% (v/v) according to the solid-liquid ratio of 1:6 (g/ml) and extract three yellow ginger powder, obtain carbon source yellow ginger saponin(e, be applied in seed activation substratum and fermention medium.
(2) inoculate Aspergillus awamori and carry out microbe conversion.Seed activation culture medium culturing is inoculated in 7 days by screening the Aspergillus awamori obtained, seed activation substratum forms: yellow ginger saponin(e 3g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 1.5g/L, bitter salt 1g/L, Repone K 1g/L, seven ferric sulfate hydrate 0.02g/L, agar powder 20g/L, pH7.5, sterilizing 15min under 121 DEG C of high pressure steam.
(3) ferment in shaking table mode.Fermentation condition is the fermention medium of the bottled 90ml of 500ml taper, and inoculum size is 6% by volume, culture temperature 32 DEG C, shaking speed 200r/min, cultivates 7 days.Fermention medium forms: yellow ginger saponin(e 3g/L, SODIUMNITRATE 3g/L, potassium primary phosphate 1.5g/L, bitter salt 1g/L, Repone K 1g/L, seven ferric sulfate hydrate 0.02g/L, pH7.5, sterilizing 15min under 121 DEG C of high pressure steam.
(4) fermentation liquor centrifuging and taking precipitation, adds organic solvent and utilizes sonic oscillation to carry out extraction diosgenin.Be that 1:3 ratio adds anhydrous methanol in yellow ginger saponin(e fermented liquid and anhydrous methanol volume ratio, extract in the mode of sonic oscillation, ultrasonic power is 100W, and vibration rotating speed 200r/min, extraction time 2h, obtains yellow ginger diosgenin.
Embodiment 3
Microbe transformation method prepares a method for yellow ginger diosgenin, comprises the following steps:
(1) in seed activation substratum and fermention medium, carbon source is yellow ginger saponin(e, with response phase method optimization obtain yellow ginger saponin(e the optimised process of extraction.Take ethanol 200r/min shaker water bath at 75 DEG C that 10g yellow ginger powder adds 70% (v/v) according to the solid-liquid ratio of 1:4 (g/ml) and extract three yellow ginger powder, obtain carbon source yellow ginger saponin(e, be applied in seed activation substratum and fermention medium.
(2) inoculate Aspergillus awamori and carry out microbe conversion.The Aspergillus awamori that screening obtains is inoculated in seed seed activation medium and cultivates 5 days, seed activation substratum forms: yellow ginger saponin(e 4g/L, peptone 6g/L, potassium primary phosphate 2g/L, bitter salt 2g/L, Repone K 2g/L, seven ferric sulfate hydrate 0.03g/L, agar powder 18g/L, pH7.0, sterilizing 15min under 121 DEG C of high pressure steam.
(3) ferment in shaking table mode.Fermentation condition is the fermention medium of the bottled 80ml of 500ml taper, and inoculum size is 7% by volume, culture temperature 35 DEG C, shaking speed 190r/min, cultivates 7 days.Fermention medium forms: yellow ginger saponin(e 4g/L, peptone 6g/L, potassium primary phosphate 2g/L, bitter salt 2g/L, Repone K 2g/L, seven ferric sulfate hydrate 0.03g/L, pH7.0, sterilizing 15min under 121 DEG C of high pressure steam.
(4) fermentation liquor centrifuging and taking precipitation, adds organic solvent and utilizes sonic oscillation to carry out extraction diosgenin.Be that 1:3 ratio adds anhydrous methanol in yellow ginger saponin(e fermented liquid and anhydrous methanol volume ratio, extract in the mode of sonic oscillation, ultrasonic power is 100W, and vibration rotating speed 200r/min, extraction time 2h, obtains yellow ginger diosgenin.
The liquid phase chromatogram condition of Fig. 1-Fig. 4: determined wavelength 200nm, flow velocity 0.9ml/min, sample size 20 μ l, mobile phase ratio acetonitrile: water=8:2.
The solvent dissolving reference substance and sample is methyl alcohol, and methyl alcohol has larger absorption at 200nm, as shown in Figure 1, and appearance time about 2min.By detecting, reference substance (diosgenin) has maximum absorption at 200nm place, appearance time about 11.8min (see Fig. 2).Yellow ginger alcohol extract obtains diosgenin after Aspergillus awamori microbe conversion, identical with reference substance appearance time (see Fig. 3).Yellow ginger alcohol extract, after the sulphuric acid hydrolysis of 2%, measures its dioscin content, and as shown in Figure 4, appearance time is identical with reference substance, and peak area is in table 2.
Accurate formulation 40 μ g/ml, 80 μ g/ml, 120 μ g/ml, 160 μ g/ml, 200 μ g/ml, the reference substance solution of 240 μ g/ml, according to given chromatographic condition sample introduction, obtains the corresponding relation of peak area and concentration, as shown in table 1.
Table 1 peak area and concentration relationship
Relation according to table 1 peak area and concentration draws out typical curve, in order to calculate the content (see Fig. 5) of converted product diosgenin.Wherein, standard equation y=24477x-178269, coefficient R
2=0.9991.
Yellow ginger ethanol extraction (alcohol extract), namely yellow ginger total saponins (not containing free diosgenin) measures the content of its diosgenin after the sulphuric acid hydrolysis of 2%, is 107.35mg/g; Then inoculation fermentation is carried out according to the method for example 1-3 respectively, the converted product diosgenin obtained enters HPLC and measures, content is respectively 75.86mg/g, 79.95mg/g and 74.67mg/g, through comparing with the dioscin content of alcohol extract, transformation efficiency can be obtained and be respectively 70.67%, 74.48% and 69.56% (see table 2), it can thus be appreciated that the alcohol extract through Aspergillus awamori fermentation can Efficient Conversion be diosgenin.
Table 2 Aspergillus awamori transformation efficiency calculates
Aspergillus awamori converted product (diosgenin) cubage formula=30/0.2*n/0.3/1000, alcohol extract acid hydrolysis products (diosgenin) cubage formula=30/0.05*n/1000.
30-Extraction solvent methanol usage (ml), 0.2/0.05-mensuration consumption (ml), n-concentration (μ g/ml), alcohol extract content (g) in 0.3-fermention medium, 1000-unit multiple.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; the change done under other any does not deviate from spirit of the present invention and principle, modification, substitute, combine, simplify the substitute mode that all should be equivalence, be included within protection scope of the present invention.
Claims (6)
1. microbe transformation method prepares a method for yellow ginger diosgenin, it is characterized in that:
(1) preparation of carbon source yellow ginger saponin(e;
(2) Aspergillus awamori is seeded in seed activation substratum carries out microbe conversion;
(3) ferment in shaking table mode;
(4) separating-purifying obtains yellow ginger diosgenin.
2. a kind of microbe transformation method according to claim 1 prepares the method for yellow ginger diosgenin, it is characterized in that: specifically comprise the steps:
(1) yellow ginger is washed with water totally in 45 DEG C ~ 50 DEG C oven for drying to constant weight, then pulverize 300 ~ 400 mesh sieves with powder beater and obtain yellow ginger raw material powder, add the extraction of ethanolic soln shaker water bath and obtain carbon source yellow ginger saponin(e for 2 ~ 3 times, add in seed activation substratum and fermention medium;
(2) Aspergillus awamori is seeded in seed activation substratum cultivates, obtain the Aspergillus awamori activated;
(3) be that 1% ~ 2% stroke-physiological saline solution dissolving is prepared into bacteria suspension by the Aspergillus awamori mass percent concentration that step (2) activates, be then seeded in fermention medium, ferment in shaking table mode, obtain yellow ginger saponin(e fermented liquid;
(4) fermentation liquor centrifuging and taking precipitation, adds organic solvent and utilizes sonic oscillation to extract, obtain yellow ginger diosgenin.
3. a kind of microbe transformation method according to claim 2 prepares the method for yellow ginger diosgenin, it is characterized in that: described in step (1), the solid-liquid ratio of yellow ginger raw material powder and ethanolic soln is 1:4 ~ 1:7 g/ml; The concentration of volume percent of described ethanolic soln is 70% ~ 80%; Described shaker water bath Extracting temperature is 70 DEG C ~ 80 DEG C, and rotating speed is 180 ~ 200r/min.
4. a kind of microbe transformation method according to claim 1 prepares the method for yellow ginger diosgenin, it is characterized in that: the described seed activation substratum composition of step (2): carbon source yellow ginger saponin(e 2-4g/L, peptone 6-8g/L or SODIUMNITRATE 3-5g/L, potassium primary phosphate 1-3g/L, 7H
2o magnesium sulfate 0.5-1.5g/L, Repone K 0.5-1.5g/L, 7H
2o ferric sulfate 0.01-0.03g/L, agar powder 18-22g/L, pH6.5-7.5; Described cultivated days is 5 ~ 7 days.
5. a kind of microbe transformation method according to claim 1 prepares the method for yellow ginger diosgenin, it is characterized in that: the fermentation condition described in step (3) is the fermention medium of the bottled 80-100ml of taper, inoculum size is 5%-8% by volume, culture temperature 28-35 DEG C, shaking speed 180-200r/min, cultivates 5-7 days; Described fermention medium composition: carbon source yellow ginger saponin(e 2-4g/L, peptone 6-8g/L or SODIUMNITRATE 3-5g/L, potassium primary phosphate 1-3g/L, bitter salt 0.5-1.5g/L, Repone K 0.5-1.5g/L, seven ferric sulfate hydrate 0.01-0.03g/L, pH6.5-7.5, sterilizing 15min under 121 DEG C of high pressure steam.
6. a kind of microbe transformation method according to claim 1 prepares the method for yellow ginger diosgenin, it is characterized in that: the described concrete grammar utilizing sonic oscillation to carry out extracting of step (4) is for adding organic solvent, extract in the mode of sonic oscillation, ultrasonic power is 200 ~ 320w, vibration rotating speed 180r/min-200r/min, extraction time 2-3h; Described organic solvent comprises anhydrous methanol or dehydrated alcohol, and the volume ratio of organic solvent and yellow ginger saponin(e fermented liquid is 2:1-3:1.
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