CN103146795B - Method for producing diosgenin through microbial fermentation of peltate yam rhizome-yellow ginger - Google Patents
Method for producing diosgenin through microbial fermentation of peltate yam rhizome-yellow ginger Download PDFInfo
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- CN103146795B CN103146795B CN201210577968.1A CN201210577968A CN103146795B CN 103146795 B CN103146795 B CN 103146795B CN 201210577968 A CN201210577968 A CN 201210577968A CN 103146795 B CN103146795 B CN 103146795B
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- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 title claims abstract description 62
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 title claims abstract description 58
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 title claims abstract description 58
- 230000000813 microbial effect Effects 0.000 title claims abstract description 10
- 238000004519 manufacturing process Methods 0.000 title abstract description 12
- 244000281702 Dioscorea villosa Species 0.000 title abstract 3
- 238000000855 fermentation Methods 0.000 title description 7
- 230000004151 fermentation Effects 0.000 title description 7
- 244000062245 Hedychium flavescens Species 0.000 claims abstract description 38
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 claims abstract description 37
- 239000000843 powder Substances 0.000 claims abstract description 31
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- 239000002002 slurry Substances 0.000 claims abstract description 22
- 239000008399 tap water Substances 0.000 claims abstract description 20
- 235000020679 tap water Nutrition 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 14
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 12
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 12
- 239000012138 yeast extract Substances 0.000 claims abstract description 12
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000004202 carbamide Substances 0.000 claims abstract description 11
- 238000002425 crystallisation Methods 0.000 claims abstract description 10
- 238000003756 stirring Methods 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 31
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- VNONINPVFQTJOC-ZGXDEBHDSA-N dioscin Chemical compound O([C@@H]1[C@@H](CO)O[C@H]([C@@H]([C@H]1O)O[C@H]1[C@@H]([C@H](O)[C@@H](O)[C@H](C)O1)O)O[C@@H]1CC2=CC[C@H]3[C@@H]4C[C@H]5[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@@H]([C@]1(OC[C@H](C)CC1)O5)C)[C@@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@H]1O VNONINPVFQTJOC-ZGXDEBHDSA-N 0.000 claims description 19
- HDXIQHTUNGFJIC-UHFFFAOYSA-N (25R)-spirost-5-en-3beta-ol 3-O-<O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside> Natural products O1C2(OCC(C)CC2)C(C)C(C2(CCC3C4(C)CC5)C)C1CC2C3CC=C4CC5OC1OC(CO)C(O)C(O)C1OC1OC(C)C(O)C(O)C1O HDXIQHTUNGFJIC-UHFFFAOYSA-N 0.000 claims description 18
- VNONINPVFQTJOC-RXEYMUOJSA-N Collettiside III Natural products O([C@@H]1[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O2)[C@H](CO)O[C@@H]1O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]5[C@H](C)[C@@]6(O[C@H]5C4)OC[C@H](C)CC6)CC3)CC=2)CC1)[C@H]1[C@H](O)[C@H](O)[C@@H](O)[C@H](C)O1 VNONINPVFQTJOC-RXEYMUOJSA-N 0.000 claims description 18
- CJNUQCDDINHHHD-APRUHSSNSA-N dioscin Natural products C[C@@H]1CC[C@@]2(OC1)O[C@H]3C[C@H]4[C@@H]5CC=C6C[C@H](CC[C@@H]6[C@H]5CC[C@]4(C)[C@H]3[C@@H]2C)O[C@@H]7O[C@H](CO)[C@@H](O[C@@H]8O[C@@H](C)[C@H](O)[C@@H](O)[C@H]8O)[C@H](O)[C@H]7O[C@@H]9O[C@@H](C)[C@H](O)[C@@H](O)[C@H]9O CJNUQCDDINHHHD-APRUHSSNSA-N 0.000 claims description 18
- VNONINPVFQTJOC-UHFFFAOYSA-N polyphyllin III Natural products O1C2(OCC(C)CC2)C(C)C(C2(CCC3C4(C)CC5)C)C1CC2C3CC=C4CC5OC(C(C1O)OC2C(C(O)C(O)C(C)O2)O)OC(CO)C1OC1OC(C)C(O)C(O)C1O VNONINPVFQTJOC-UHFFFAOYSA-N 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 230000009466 transformation Effects 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 8
- 230000000737 periodic effect Effects 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 230000007062 hydrolysis Effects 0.000 claims description 7
- 238000006460 hydrolysis reaction Methods 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012531 culture fluid Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 238000004809 thin layer chromatography Methods 0.000 claims description 6
- 238000005273 aeration Methods 0.000 claims description 5
- 230000035614 depigmentation Effects 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000012262 fermentative production Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 claims description 4
- 239000007921 spray Substances 0.000 claims description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000004744 fabric Substances 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 238000000703 high-speed centrifugation Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000012807 shake-flask culturing Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 235000013573 potato product Nutrition 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 10
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- 150000007949 saponins Chemical class 0.000 abstract description 6
- 238000003912 environmental pollution Methods 0.000 abstract description 3
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- 238000004321 preservation Methods 0.000 abstract description 2
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- 239000001397 quillaja saponaria molina bark Substances 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for producing diosgenin through microbial conversion of peltate yam rhizome-yellow ginger. Yellow ginger dry powder and tap water are mixed according to a massic volume ratio of 1:5-12, urea which is 4.0% of mass of the yellow ginger dry powder, yeast extract which is 1.0% of mass of the yellow ginger dry powder and inorganic salt which is 0.2% of volume of the tap water are supplemented to prepare conversion slurry, the conversion slurry is introduced in bacillus bacterial strain XBT 2011 seed liquid which is 5% of the volume of the tap water, under the condition that the culture temperature is 40 DEG C, the pH is controlled to be about 6.0, the stirring speed is 300-400rpm and the ventilation condition is 2.5-4.0l/min, the conversion is 56-72hrs, conversion slurry is filtered and dried to obtain hydrolysate, the hydrolysate is extracted for 12-16hrs by using 120# gasoline, the volume of the 120# gasoline is one time of that of the hydrolysate, and diosgenin end products are obtained through repeated crystallization. A preservation number of the bacterial strain XBT 2011 is CGMCCNo.6301. The dry weight of the hydrolysate is 28-33% of that of the added yellow ginger dry powder, total saponins of the peltate yam rhizome is approximately converted into diosgenin completely, handling capacity is big, no sugar contains, the pH is close to neutral, and environmental pollution is reduced.
Description
Technical field
The present invention relates to a kind of method of preparing diosgenin, particularly a kind of method of being produced diosgenin by Rhizome of Peltate Yam-yellow ginger through microbial transformation.Belong to biological extraction technical field.
Background technology
From yellow ginger, extracting the most frequently used method of diosgenin (saponin) is exactly acid-hydrolysis method, this production technique the earliest exist hydrolysis not exclusively, the problems such as the huge and serious environment pollution of yield low (yield is generally only in 1.7% left and right), wastewater flow rate.The method that spontaneous fermentation after each enterprise's employing at present improves and acid-hydrolysis method are coupled.Spontaneous fermentation, by the enzymolysis of yellow ginger self endogenous enzyme, makes a part of furan Zi alcohol saponin be converted into spiral shell Zi alcohol saponin, and the latter Geng Yi is converted into diosgenin.Therefore this method being coupled is produced saponin, and the direct acid-hydrolysis method of its productivity ratio can improve 10% ~ 25%.But owing to there being multiple-microorganism participate in fermentation and transform, system endoenzyme kind complexity, along with the prolongation of fermentation time, may generate sapogenin ketone and impurity in extract is increased, melting point depression, and still have that acid hydrolysis is incomplete, yield is low (yield is generally only in 1.8% left and right), wastewater flow rate is huge and the problems such as serious environment pollution.
For solving appeal Current Situation, domestic Duo Jia R&D institution selects to utilize the method for microorganism fermentation to carry out the microbial transformation of diosgenin.The microbial strains wherein using is mainly taking mould as main, according to incompletely statistics, these mould bacterial classifications cover Coriolus Versicolors (
coriolus versicolor), Cunninghammella (
cunnonghamella), Mucor (
mucor), aspergillus niger (
aspergillus niger), flavus (
aspergillus flavus), aspergillus oryzae (
aspergillus oryzae), terreus (
aspergillus terreus), Aspergillus fumigatus (
aspergillus fumigatus) and Penicillium (
penicillium) etc. the fungal strain of multiple different genus or kind.Utilize mould to produce enzyme and carry out dioscin hydrolysis, its inferior position is that the hydrolysis specificity of enzyme is poor, and the growth cycle of mould itself has also determined the chronicity (report mostly is 5-7 at present) of fermentation period, the polysaccharide component palliating degradation degree of yellow ginger is low in addition, increase follow-up extraction difficulty, make production cost higher, be therefore difficult to be applied to suitability for industrialized production.For this reason, people propose again acidic hydrolysis, after yellow ginger pulverizing is sized mixing, add a certain amount of amylase, cellulase, polygalacturonase etc. to carry out enzymolysis, rear access mould seed liquor is carried out liquid fermenting, produces diosgenin. and this method is utilized plurality of enzymes liquid degraded starch, the polysaccharide components such as Mierocrystalline cellulose, " parcel " and " shielding " effect that releasing starch etc. produces dioscin, more can fully be hydrolyzed it.But due to a large amount of zymins that use, production cost is increased substantially, acidic hydrolysis is greatly differed from each other from suitability for industrialized production.
In a word, although microbe transformation method production diosgenin represents a new direction of saponin industry development, but due to the simplification (only limiting to fungal strain) of strain screening kind and the problem such as the enzymolysis efficiency of experimental strain is low, make this method slowly fail to break through the bottleneck in laboratory and enter industrial applications.Therefore how to filter out enzymolysis dioscin efficiency high, the microorganism strains of processing power strong (blending ratio of ginger powder and water is high) and the yellow ginger polysaccharide component of simultaneously degrading (lowering subsequent extracted cost) is the key point that microorganism enzymolysis conversion method is produced diosgenin.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, solve the bottleneck problem of microbe transformation method production diosgenin, the present invention aims to provide a kind of method of being produced diosgenin by Rhizome of Peltate Yam-yellow ginger through microbial transformation.Completely, the efficiency of producing diosgenin is high in the method hydrolysis, and processing power is strong and cost is low, is expected to realize large-scale industrial production.
In order to achieve the above object, the technical scheme that the present invention takes is: a kind of by Rhizome of Peltate Yam-yellow ginger the method through microorganism fermentative production diosgenin, it is characterized in that: yellow ginger dry powder and tap water are mixed in the ratio of mass volume ratio 1:5 ~ 12, and fill into into the urea of yellow ginger dry powder quality 4.0%, for the yeast extract of yellow ginger dry powder quality 1.0% be the inorganic salt of tap water volume 0.2%, be made into conversion slurries, access volume be about tap water volume 5% bacillus (
baccilus) strain X BT2011 seed liquor, 40 DEG C of culture temperature, control pH 6.0, stirring velocity is 300 ~ 400rpm, aeration condition is to transform 56 ~ 72hrs under the culture condition of 2.5 ~ 4.0 l/min, transforms slurries after filtration, the dry hydrolyzate that obtains, 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid for hydrolyzate, obtain diosgenin finished product through periodic crystallisation; Described rpm is rev/min, and hrs is hour; Described bacillus (
baccilus) preserving number of strain X BT2011: CGMCCNo.6301.
In conversion process, from fermentor tank, get 5ml conversion fluid every 6hrs and detect diosgenin conversion situation; To the water-saturated n-butanol reagent that adds 2 times of liquid sample volumes in liquid sample, high speed centrifugation 5min after ultrasonic echography 20min, get supernatant liquor in crucible in 100 DEG C of oven dry, again to the methanol solution of the liquid sample volume that doubles in crucible, after repeatedly dissolving, filter, get appropriate filtrate point sample in thin layer chromatography board, the chloroform/methanol/water developping agent that is 7: 3: 0.5 by volume ratio launches, spray 10ml mass percent concentration 10% H
2sO
4solution heating colour developing, contrasts with the pre-sapogenin mark of potato product, observes hydrolysis degree and the diosgenin transforming degree of total dioscin; Cultivate slurries and filter through industrial filter cloth, obtain hydrolyzate in 85 DEG C of oven dry, 120 long-pending ﹟ gasoline extraction 12-16hrs of about monoploid for hydrolyzate, and add industrial gac depigmentation, obtain diosgenin finished product through periodic crystallisation.
Gained hydrolyzate dry weight in the method, by being added yellow ginger dry powder dry weight 28～33%, diosgenin yield is 2.7～3.05%.
Described inorganic salt are N
acl, MgSO
4, k
2hPO
4, FeSO
4and CaCl
2mixture.
Described inorganic salt are to be divided into 0.1% N by quality volume percent
acl, 0.05%MgSO
4, 0.05%k
2hPO
4, 0.005%FeSO
4and 0.001%CaCl
2composition.
Being prepared as of described seed liquor got 5ml cell density 10
8～10
9/ ml's
bacillus aryabhattaixBT2011 suspension inoculation, in 150ml seed culture fluid, at 40 DEG C, is cultivated 14hrs under the shake-flask culture condition of 200rpm and is made; Wherein said seed culture fluid is made up of by the long-pending per-cent of following mass body following composition: yellow ginger dry powder 5%, urea 0.3%, yeast extract 0.1%, N
acl 0.1%, MgSO
40.05%, k
2hPO
40.05%, FeSO
40.005%, CaCl
20.001%.
The advantage of this technology is: for thoroughly changing the production status of saponin industry, through a large amount of arduous screening operations, from 102 different soil samples, enrichment screening repeatedly, obtains 450 strain candidate strain from nearly 10000 strain bacterial strains, then sieves again and finally selected 3 strain desirable strain through transformation efficiency.Wherein a strain through be accredited as bacillus (
baccilus) strain X BT2011, in preservation on the 27th in 06 month in 2012, depositary institution's title: Institute of Microorganism, Academia Sinica, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101; Deposit number: CGMCC No.6301, Classification And Nomenclature: A Shi genus bacillus (
bacillus aryabhattai).
Through approximately 40 5L fermentor tank (Sartorius, Germany) experiment, yellow ginger dry powder and tap water are mixed in the ratio of mass volume ratio 1:5 ~ 12, and fill into into the urea of yellow ginger dry powder quality 4.0%, for the yeast extract of yellow ginger dry powder quality 1.0% be the inorganic salt of tap water volume 0.2%, be made into conversion slurries, access volume be about tap water volume 5% bacillus (
baccilus) strain X BT2011 seed liquor, 40 DEG C of culture temperature, control pH 6.0 left and right, stirring velocity is 300 ~ 400rpm, aeration condition is to transform 56 ~ 72hrs under the culture condition of 2.5 ~ 4.0 l/min, transforms slurries after filtration, the dry hydrolyzate that obtains, 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid for hydrolyzate, obtain diosgenin finished product through periodic crystallisation.Gained hydrolyzate dry weight, by being added yellow ginger dry powder dry weight 28～33%, diosgenin yield is 2.7～3.05%.This processing method can make total dioscin approach and change into diosgenin completely, treatment capacity reaches industrialized level, and the waste liquid amount producing is about 1/3 of current industrialization status, and not containing sugar, pH, close to neutrality, can reduce environmental pollution to greatest extent.
Compare with the patent CN101012474B of nearest bulletin, the present invention selects bacterial isolates to transform bacterial classification as dioscin at home for the first time, not only enzymolysis total dioscin is complete for this bacterial strain, transform production total dioscin unit efficiency high, its main advantage is farthest to improve yellow ginger treatment capacity (yellow ginger dry powder and water mix taking mass volume ratio as 1:5), approach industrial actual treatment level, nearly 7 times of the yellow ginger feed concentration mentioned of patent CN101012474B, thereby can reduce conversion cost, reduce to greatest extent the growing amount of waste water, be expected to realize large-scale industrial production.
Below by the specific embodiment providing, the present invention can be further clearly described, but not as a limitation of the invention.
Embodiment
A kind of by Rhizome of Peltate Yam-yellow ginger the method through microorganism fermentative production diosgenin, it is characterized in that: yellow ginger dry powder and tap water are mixed in the ratio of mass volume ratio 1:5 ~ 12, and fill into into the urea of yellow ginger dry powder quality 4.0%, for the yeast extract of yellow ginger dry powder quality 1.0% be the inorganic salt of tap water volume 0.2%, be made into conversion slurries, access volume be tap water volume 5% bacillus (
baccilus) strain X BT2011 seed liquor, 40 DEG C of culture temperature, control pH 6.0, stirring velocity is 300 ~ 400rpm, aeration condition is to transform 56 ~ 72hrs under the culture condition of 2.5 ~ 4.0 l/min, transforms slurries after filtration, the dry hydrolyzate that obtains, 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid for hydrolyzate, obtain diosgenin finished product through periodic crystallisation; Described rpm is rev/min, and hrs is hour; Described bacillus (
baccilus) preserving number of strain X BT2011: CGMCCNo.6301.
Embodiment 1
The preparation of seed liquor
Get 5ml cell density 10
8～10
9/ ml's
bacillus aryabhattaixBT2011 suspension inoculation, in 150ml seed culture fluid, at 40 DEG C, is cultivated 14hrs under the shake-flask culture condition of 200rpm and is made seed liquor.Wherein seed culture fluid is made up of by the long-pending per-cent of following mass body following composition: yellow ginger dry powder 5%, urea 0.3%, yeast extract 0.1%, N
acl 0.1%, MgSO
40.05%, k
2hPO
40.05%, FeSO
40.005%, CaCl
20.001%.
Transform the preparation of slurries
Add 500g yellow ginger dry powder and 3000mL tap water in 5L fermentor tank, and add urea 20g, yeast extract 5.0g, N
acl 3.0g, MgSO
41.5g, k
2hPO
41.5g, FeSO
40.15g, CaCl
20.03g composition, wherein inorganic salt are tap water volume 0.2%, adjust pH7.0 ± 0.2.In 121 DEG C, sterilizing 15min under 0.1Mpa pressure condition (minute).
The conversion of diosgenin
In sterilized conversion slurries, access above-mentioned seed liquor 150ml, in 40 DEG C, under 350rpm rotating speed and 3.0 L/min aeration conditions, cultivate.In experimentation, intermittently stream adds 20% ammoniacal liquor control pH in 6.0 left and right, and stream adds about 5ml defoamer control foam generation simultaneously.Cultivate conversion 64hrs and put tank after diosgenin transforms completely.Cultivate slurries filter through industrial filter cloth, obtain hydrolyzate dry weight 155.8g in 85 DEG C of oven dry, by interpolation yellow ginger dry powder dry weight 31.1%.120 long-pending ﹟ gasoline extraction 12-16hrs of about monoploid for hydrolyzate, and add industrial gac depigmentation, obtain diosgenin finished product 14.5g through periodic crystallisation, and diosgenin finished product yield is that to measure its dioscin content be 95.6% to 2.9%, RP-HpLC.Described rpm is rev/min, and hrs is hour; Described bacillus (
baccilus) preserving number of strain X BT2011: CGMCCNo.6301.
In conversion process, from fermentor tank, get 5ml conversion fluid every 6hrs and detect diosgenin conversion situation.To the water-saturated n-butanol reagent that adds 2 times of liquid sample volumes in liquid sample, high speed centrifugation 5min after ultrasonic echography 20min, get supernatant liquor in crucible in 100 DEG C of oven dry, again to the methanol solution of the liquid sample volume that doubles in crucible, after repeatedly dissolving, filter, get appropriate filtrate point sample in thin layer chromatography board, the chloroform/methanol/water developping agent that is 7: 3: 0.5 by volume ratio launches, and sprays about 10ml mass percent concentration 10% H
2sO
4solution heating colour developing, contrasts with diosgenin mark product, observes hydrolysis degree and the diosgenin transforming degree of total dioscin.The demonstration of thin-layer chromatography result, along with the conversion of total dioscin, total saponins color spot shoals gradually to such an extent as to disappears, and the color spot of diosgenin colour developing is simultaneously further obvious, can make the color spot completely dissolve of total dioscin after conversion 64hrs.
The detection of diosgenin structure
Get the ultrasonic assisted extraction of 0.5g said hydrolyzed thing 2ml chloroform, will after extracting liquid filtering, get appropriate filtrate point sample in thin layer chromatography board, the chloroform/acetone developping agent that is 9:1 by volume ratio launches, and sprays about 10ml mass percent concentration 10% H
2sO
4solution heating colour developing, contrasts with the color spot of mark product diosgenin, and result shows that conversion diosgenin is in full accord with the Rf value of mark product diosgenin color spot.
Take a morsel sample after 80 DEG C of oven dry, grind compressing tablet sample preparation with KBr, with the infared spectrum of determination of infrared spectroscopy sample, sweep limit 4,000 one 400cm
-1.Experimental result shows that diosgenin standard substance are consistent with diosgenin finished product infrared spectrogram.A small amount of sample is packed in crucible into AL
20
3crucible is as reference crucible.Adopt high pure nitrogen as protection gas and sweep gas, both are adjusted into 30mL/min by flow, and pyrolysis oven temperature rise rate is 5 DEG C/min.Sample rises to 250 DEG C from room temperature, degradation production is purged out pyrolysis oven by high pure nitrogen simultaneously, record thermogravimetric curve and differential scanning calorimetric curve, can find out from means of differential scanning calorimetry (DSC) curve, diosgenin finished product melting temperature is 200 ~ 203 DEG C.Can confirm that according to above detected result yellow ginger total dioscin is after bacterial isolates XBT2011 transforms, its converted product is the pre-sapogenin of potato.
The measuring method (RPLC RP-HpLC) of Determination of Diosgenin: Agilent1100 highly effective liquid phase chromatographic system; Chromatographic column: ZorbaxC18 (150mm × 4.6mmi.d.), moving phase: methanol/water=90/10 (v/v), and detect wavelength: 210nm, flow velocity: 1mL/min, column temperature is normal temperature.
Embodiment 2
Basic identical with embodiment 1, difference is:
Add 600g yellow ginger dry powder and 3000mL tap water in 5L fermentor tank, add urea 25g, yeast extract 6.0g, N
acl 3.0g, MgSO
41.5g, k
2hPO
41.5g, FeSO
40.15g, CaCl
2the one-tenth such as 0.03g are distributed into conversion slurries, and wherein inorganic salt are tap water volume 0.2%.Cultivate the XBT2011 bacterial strain seed liquor 200ml of 14hrs to the conversion slurries access of sterilizing, at 40 DEG C, 400rpm, 3.5 l/min, transform 72hrs under the culture condition of control pH 6.0.Transform slurries after filtration, dry, finally obtain hydrolyzate dry weight 198.7g, by interpolation yellow ginger dry powder dry weight 33.1%.120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid for hydrolyzate, and add industrial gac depigmentation, obtain diosgenin finished product 16.5g through periodic crystallisation, and diosgenin yield is that to measure its dioscin content be 95.6% to 2.7%, RP-HpLC.
Embodiment 3
Basic identical with embodiment 12, difference is:
Add the dry yellow ginger powder of 250g and 3000mL water in 5L fermentor tank, add urea 10g, yeast extract 2.5g, N
acl 3.0g, MgSO
41.5g, k
2hPO
41.5g, FeSO
40.15g, CaCl
2the one-tenth such as 0.03g are distributed into conversion slurries, and wherein inorganic salt are tap water volume 0.2%.Cultivate the XBT2011 bacterial strain seed liquor 200ml of 14hrs to the conversion slurries access of sterilizing, in 40 DEG C, 300rpm, 2.5 l/min, transform 56hrs under the culture condition of control pH 6.2.Cultivate slurries after filtration, the dry hydrolyzate dry weight 70.5g that obtains, by interpolation yellow ginger dry powder dry weight 28.2%.120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid for hydrolyzate, and add industrial gac depigmentation, obtain diosgenin finished product 7.63g through crystallization, and soap dioscin yield is that to measure its dioscin content be 96.2% to 3.05%, RP-HpLC.Thin-layer chromatography result shows that the transformation efficiency of total dioscin is 100%.
Described yeast extract is taking high protein bread yeast or cereuisiae fermentum as raw material, the biological culture base product of the nutritive ingredients such as a kind of rich in proteins of making through self-dissolving, enzymolysis, the technique such as concentrated, dry, amino acid, peptide, polypeptide, nucleic acid, electrolytes and minerals, belongs to known technology and is not described in detail.
Gained hydrolyzate dry weight in this technique, by being added yellow ginger dry powder dry weight 28～33%, diosgenin yield is 2.7～3.05%.This processing method can make total dioscin approach and change into diosgenin completely, treatment capacity reaches industrialized level, and the waste liquid amount producing is about 1/3 of current industrialization status, and not containing sugar, pH, close to neutrality, can reduce environmental pollution to greatest extent.
The part that the present embodiment does not describe in detail and english abbreviation belong to the common practise of the industry, can search, here not narration one by one on the net.
Claims (5)
- One kind by Rhizome of Peltate Yam-yellow ginger the method through microorganism fermentative production diosgenin, it is characterized in that: yellow ginger dry powder and tap water are mixed in the ratio of mass volume ratio 1:5 ~ 12, and fill into into the urea of yellow ginger dry powder quality 4.0%, for the yeast extract of yellow ginger dry powder quality 1.0% be the inorganic salt of tap water volume 0.2%, be made into conversion slurries, access volume be about tap water volume 5% bacillus ( baccilus) strain X BT2011 seed liquor, 40 DEG C of culture temperature, control pH 6.0, stirring velocity is 300 ~ 400rpm, aeration condition is to transform 56 ~ 72hrs under the culture condition of 2.5 ~ 4.0 l/min, transforms slurries after filtration, the dry hydrolyzate that obtains, 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid for hydrolyzate, obtain diosgenin finished product through periodic crystallisation; Described rpm is rev/min, and hrs is hour; Described bacillus ( baccilus) preserving number of strain X BT2011: CGMCCNo.6301; Being prepared as of described seed liquor got 5ml cell density 10 8～10 9/ ml's bacillus aryabhattaixBT2011 suspension inoculation, in 150ml seed culture fluid, at 40 DEG C, is cultivated 14hrs under the shake-flask culture condition of 200rpm and is made; Wherein said seed culture fluid is made up of by the long-pending per-cent of following mass body following composition: yellow ginger dry powder 5%, urea 0.3%, yeast extract 0.1%, N acl 0.1%, MgSO 40.05%, k 2hPO 40.05%, FeSO 40.005%, CaCl 20.001%.
- 2. a kind of method of being produced diosgenin by Rhizome of Peltate Yam-yellow ginger through microbial transformation according to claim 1, is characterized in that: in conversion process, get 5ml conversion fluid detect diosgenin conversion situation every 6hrs from fermentor tank; To the water-saturated n-butanol reagent that adds 2 times of liquid sample volumes in liquid sample, high speed centrifugation 5min after ultrasonic echography 20min, get supernatant liquor in crucible in 100 DEG C of oven dry, again to the methanol solution of the liquid sample volume that doubles in crucible, after repeatedly dissolving, filter, get appropriate filtrate point sample in thin layer chromatography board, the chloroform/methanol/water developping agent that is 7: 3: 0.5 by volume ratio launches, spray 10ml mass percent concentration 10% H 2sO 4solution heating colour developing, contrasts with the pre-sapogenin mark of potato product, observes hydrolysis degree and the diosgenin transforming degree of total dioscin; Cultivate slurries and filter through industrial filter cloth, obtain hydrolyzate in 85 DEG C of oven dry, 120 long-pending ﹟ gasoline extraction 12-16hrs of about monoploid for hydrolyzate, and add industrial gac depigmentation, obtain diosgenin finished product through periodic crystallisation.
- 3. a kind of method of being produced diosgenin by Rhizome of Peltate Yam-yellow ginger through microbial transformation according to claim 1 and 2, it is characterized in that: gained hydrolyzate dry weight in the method, by being added yellow ginger dry powder dry weight 28～33%, diosgenin yield is 2.7～3.05%.
- 4. a kind of method of being produced diosgenin by Rhizome of Peltate Yam-yellow ginger through microbial transformation according to claim 1, is characterized in that: described inorganic salt are N acl, MgSO 4, k 2hPO 4, FeSO 4and CaCl 2mixture.
- 5. according to a kind of method of being produced diosgenin by Rhizome of Peltate Yam-yellow ginger through microbial transformation described in claim 1 or 4, it is characterized in that: described inorganic salt are to be divided into 0.1% N by quality volume percent acl, 0.05%MgSO 4, 0.05%k 2hPO 4, 0.005%FeSO 4and 0.001%CaCl 2composition.
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| CN103497987B (en) * | 2013-09-16 | 2015-02-25 | 陕西科技大学 | Clean production method of yam diosgenin |
| CN104109644A (en) * | 2014-05-29 | 2014-10-22 | 华中科技大学 | Bacillus sp. and its use in natural product extraction |
| CN106834169B (en) * | 2017-01-12 | 2019-09-24 | 华中科技大学 | A kind of method that microorganism mixed fermentation prepares yellow ginger saponin(e |
| CN107177662A (en) * | 2017-07-12 | 2017-09-19 | 西北农林科技大学 | The fermentation preparation of Chinese yam saponin |
| CN107190042B (en) * | 2017-07-12 | 2020-06-02 | 西北农林科技大学 | Method for preparing diosgenin by fermentation of peltate yam endophyte |
| CN108070631A (en) * | 2017-12-29 | 2018-05-25 | 佛山科学技术学院 | A kind of method of aspergillus oryzae conversion iron rod yam production diosgenin |
| CN110596299A (en) * | 2019-10-23 | 2019-12-20 | 葵花药业集团（襄阳）隆中有限公司 | Thin-layer chromatography identification method for asparagus cochinchinensis decoction pieces in Erdong decoction formula |
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