CN103146795A - 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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- CN103146795A CN103146795A CN2012105779681A CN201210577968A CN103146795A CN 103146795 A CN103146795 A CN 103146795A CN 2012105779681 A CN2012105779681 A CN 2012105779681A CN 201210577968 A CN201210577968 A CN 201210577968A CN 103146795 A CN103146795 A CN 103146795A
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- diosgenin
- yellow ginger
- dry powder
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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 63
- 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 59
- 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 59
- 230000000813 microbial effect Effects 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 14
- 238000000855 fermentation Methods 0.000 title claims description 11
- 230000004151 fermentation Effects 0.000 title claims description 11
- 244000281702 Dioscorea villosa Species 0.000 title abstract 3
- 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 29
- 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 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 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 19
- 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 19
- 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
- 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 19
- 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 19
- 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
- 238000001914 filtration Methods 0.000 claims description 10
- 230000009466 transformation Effects 0.000 claims description 10
- 230000000737 periodic effect Effects 0.000 claims description 8
- 230000007062 hydrolysis Effects 0.000 claims description 7
- 238000006460 hydrolysis reaction Methods 0.000 claims description 7
- 239000000203 mixture Substances 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
- 238000001035 drying Methods 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
- 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
- 229930182490 saponin Natural products 0.000 abstract description 8
- 235000017709 saponins Nutrition 0.000 abstract description 8
- 150000007949 saponins Chemical class 0.000 abstract description 6
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 230000008025 crystallization Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 abstract 2
- 239000000413 hydrolysate Substances 0.000 abstract 2
- 239000007795 chemical reaction product Substances 0.000 abstract 1
- 230000007935 neutral effect Effects 0.000 abstract 1
- 238000009423 ventilation Methods 0.000 abstract 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 6
- 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
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- -1 alcohol saponin Chemical class 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000002351 wastewater Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241001225321 Aspergillus fumigatus Species 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 2
- 240000006439 Aspergillus oryzae Species 0.000 description 2
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000222355 Trametes versicolor Species 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229940091771 aspergillus fumigatus Drugs 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
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- 239000007789 gas Substances 0.000 description 2
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- 238000002844 melting Methods 0.000 description 2
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- 238000000197 pyrolysis Methods 0.000 description 2
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- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 241001465318 Aspergillus terreus Species 0.000 description 1
- 241000976738 Bacillus aryabhattai Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
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- 241000234314 Zingiber Species 0.000 description 1
- 235000006886 Zingiber officinale Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
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- 231100000749 chronicity Toxicity 0.000 description 1
- 239000013530 defoamer Substances 0.000 description 1
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Landscapes
- 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 for preparing diosgenin, particularly a kind of method of diosgenin of being produced through microbial transformation by Rhizome of Peltate Yam-yellow ginger.Belong to the biological extraction technical field.
Background technology
Extracting the most frequently used method of diosgenin (saponin) from yellow ginger is exactly acid-hydrolysis method, this production technique the earliest exist hydrolysis not exclusively, yield low (yield is generally only in 1.7% left and right), wastewater flow rate is huge and the problems such as serious environment pollution.The method that spontaneous fermentation after each enterprise's employing improves at present and acid-hydrolysis method are coupled.Spontaneous fermentation makes a part of furan Zi alcohol saponin be converted into spiral shell Zi alcohol saponin by the enzymolysis of yellow ginger self endogenous enzyme, and latter Geng Yi is converted into diosgenin.Therefore this method that is 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 is complicated, prolongation along with fermentation time, may generate sapogenin ketone and make that in extract, impurity increases, melting point depression, and have still 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 the appeal Current Situation, domestic many R﹠D institutions select to utilize the method for microbial fermentation to carry out the microbial transformation of diosgenin.Use therein microbial strains is mainly take 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. a plurality of different genus or the fungal strain of kind.Utilize mould to produce enzyme and carry out the dioscin hydrolysis, its inferior position is that the hydrolysis specificity of enzyme is relatively 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, increased follow-up extraction difficulty, make production cost higher, therefore be difficult to be applied to suitability for industrialized production.For this reason, people propose again acidic hydrolysis, namely after the 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, releasing starch etc. more can fully be hydrolyzed it to " parcel " and " shielding " effect that dioscin produces.But due to a large amount of zymins of using, 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 is produced the new direction that diosgenin represents the saponin industry development, but due to the problem such as the enzymolysis efficiency of the simplification (only limiting to fungal strain) of strain screening kind and 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 efficient high, the microorganism strains of processing power strong (blending ratio of ginger powder and water is high) and the yellow ginger polysaccharide component of degrading simultaneously (lowering the 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 that microbe transformation method is produced diosgenin, the present invention aims to provide a kind of method of diosgenin of being produced through microbial transformation by Rhizome of Peltate Yam-yellow ginger.The method hydrolysis is complete, and the efficient of producing diosgenin is high, 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 method of diosgenin of being produced through microbial fermentation by Rhizome of Peltate Yam-yellow ginger, 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 urea into 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 the conversion slurries, the access volume be about tap water volume 5% bacillus (
Baccilus) strain X BT2011 seed liquor, 40 ℃ 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, and drying obtains hydrolyzate, hydrolyzate obtains the diosgenin finished product with 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid 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, get the 5ml conversion fluid every 6hrs and detect diosgenin conversion situation from fermentor tank; The water-saturated n-butanol reagent that adds 2 times of liquid sample volumes in the liquid sample, high speed centrifugation 5min after ultrasonic echography 20min, get supernatant liquor to the crucible in 100 ℃ of oven dry, the methanol solution of liquid sample volume again doubles in the crucible, through repeatedly filtering after dissolving, getting appropriate filtrate point sample on thin layer chromatography board, is that the chloroform/methanol/water developping agent of 7: 3: 0.5 is launched with volume ratio, spray 10ml mass percent concentration 10% H
2SO
4Solution heating colour developing with the contrast of the pre-sapogenin mark of potato product, is observed hydrolysis degree and the diosgenin transforming degree of total dioscin; Cultivate slurries and filter through industrial filter cloth, obtain hydrolyzate in 85 ℃ of oven dry, the 120 ﹟ gasoline extraction 12-16hrs that hydrolyzate amasss with about monoploid, and add industrial gac depigmentation, obtain the diosgenin finished product through periodic crystallisation.
Gained hydrolyzate dry weight in the method, by being added yellow ginger dry powder dry weight 28～33%, the 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 the quality volume percent
aCl, 0.05%MgSO
4, 0.05%k
2HPO
4, 0.005%FeSO
4And 0.001%CaCl
2Form.
Being prepared as of described seed liquor got the 5ml cell density 10
8～10
9/ ml's
Bacillus aryabhattaiThe XBT2011 suspension inoculation at 40 ℃, is cultivated 14hrs and is made in the 150ml seed culture fluid under the shake-flask culture condition of 200rpm; Wherein said seed culture fluid is comprised 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 present technique is: for thoroughly changing the production status of saponin industry, through a large amount of arduous screening operations, enrichment screening repeatedly, obtain 450 strain candidate strain from nearly 10000 strain bacterial strains, then sieve again through transformation efficiency and finally selected 3 strain desirable strain from 102 different soil samples.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: the A Shi genus bacillus (
Bacillus aryabhattai).
Process is 40 5L fermentor tank (Sartorius approximately, Germany) experiment, yellow ginger dry powder and tap water are mixed in the ratio of mass volume ratio 1:5 ~ 12, and fill into urea into 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 the conversion slurries, the access volume be about tap water volume 5% bacillus (
Baccilus) strain X BT2011 seed liquor, 40 ℃ 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, and drying obtains hydrolyzate, hydrolyzate obtains the diosgenin finished product with 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid through periodic crystallisation.Gained hydrolyzate dry weight, by being added yellow ginger dry powder dry weight 28～33%, the diosgenin yield is 2.7～3.05%.This processing method can make total dioscin approach and change into diosgenin fully, treatment capacity reaches industrialized level, and the waste liquid amount that produces is about 1/3 of present industrialization status, and does not contain sugar, pH can reduce environmental pollution to greatest extent close to neutrality.
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 the enzymolysis total dioscin is complete for this bacterial strain, transform the first efficient of production total dioscin high, its main advantage is farthest to improve yellow ginger treatment capacity (yellow ginger dry powder and water mix take mass volume ratio as 1:5), near industrial actual treatment level, nearly 7 times of the yellow ginger feed concentration mentioned of patent CN101012474B, thereby can reduce the 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 that provides, the present invention can be described clearly further, but not as a limitation of the invention.
Embodiment
A kind of method of diosgenin of being produced through microbial fermentation by Rhizome of Peltate Yam-yellow ginger, 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 urea into 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 the conversion slurries, the access volume be tap water volume 5% bacillus (
Baccilus) strain X BT2011 seed liquor, 40 ℃ 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, and drying obtains hydrolyzate, hydrolyzate obtains the diosgenin finished product with 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid 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 the 5ml cell density 10
8～10
9/ ml's
Bacillus aryabhattaiThe XBT2011 suspension inoculation at 40 ℃, is cultivated 14hrs and is made seed liquor in the 150ml seed culture fluid under the shake-flask culture condition of 200rpm.Wherein seed culture fluid is comprised 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 the 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%, transfer pH7.0 ± 0.2.In 121 ℃, the 15min that sterilizes under the 0.1Mpa pressure condition (minute).
The conversion of diosgenin
Access above-mentioned seed liquor 150ml in sterilized conversion slurries, in 40 ℃, cultivate under 350rpm rotating speed and 3.0 L/min aeration conditions.In experimentation, intermittently stream adds 20% ammoniacal liquor control pH in 6.0 left and right, and stream adds approximately 5ml defoamer control foam generation simultaneously.Cultivate conversion 64hrs and put tank after diosgenin transforms fully.Cultivate slurries and filter through industrial filter cloth, obtain hydrolyzate dry weight 155.8g in 85 ℃ of oven dry, by interpolation yellow ginger dry powder dry weight 31.1%.Hydrolyzate is with 120 long-pending ﹟ gasoline extraction 12-16hrs of about monoploid, and adds industrial gac depigmentation, obtains 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, get the 5ml conversion fluid every 6hrs and detect diosgenin conversion situation from fermentor tank.The water-saturated n-butanol reagent that adds 2 times of liquid sample volumes in the liquid sample, high speed centrifugation 5min after ultrasonic echography 20min, get supernatant liquor to the crucible in 100 ℃ of oven dry, the methanol solution of liquid sample volume again doubles in the crucible, through repeatedly filtering after dissolving, getting appropriate filtrate point sample on thin layer chromatography board, is that the chloroform/methanol/water developping agent of 7: 3: 0.5 is launched with volume ratio, and spray is 10ml mass percent concentration 10% H approximately
2SO
4Solution heating colour developing with the contrast of diosgenin mark product, is observed hydrolysis degree and the diosgenin transforming degree of total dioscin.The demonstration of thin-layer chromatography result, along with the conversion of total dioscin, the disappearance to such an extent as to the total saponins color spot shoals gradually, the color spot of diosgenin colour developing simultaneously is further obvious, can make the color spot completely dissolve of total dioscin after conversion 64hrs.
The detection of diosgenin structure
Get 0.5g said hydrolyzed thing with the ultrasonic assisted extraction of 2ml chloroform, will get appropriate filtrate point sample after extracting liquid filtering on thin layer chromatography board, the chloroform/acetone developping agent that is 9:1 with volume ratio launches, and spray is 10ml mass percent concentration 10% H approximately
2SO
4Solution heating colour developing contrasts with the color spot of mark product diosgenin, and result shows that to transform diosgenin in full accord with the Rf value of mark product diosgenin color spot.
Take a morsel sample after 80 ℃ of oven dry, grind the compressing tablet sample preparation with KBr, with the infared spectrum of determination of infrared spectroscopy sample, sweep limit 4,000 one 400cm
-1Experimental result shows that the diosgenin standard substance are consistent with diosgenin finished product infrared spectrogram.A small amount of sample is packed in crucible, AL
20
3Crucible is as the reference crucible.Adopt high pure nitrogen as protection gas and sweep gas, both are adjusted into 30mL/min by flow, and the pyrolysis oven temperature rise rate is 5 ℃/min.Sample rises to 250 ℃ from room temperature, and high pure nitrogen purges out pyrolysis oven with degradation production simultaneously, records thermogravimetric curve and differential scanning calorimetric curve, can find out from means of differential scanning calorimetry (DSC) curve, and diosgenin finished product melting temperature is 200 ~ 203 ℃.Can confirm the yellow ginger total dioscin after bacterial isolates XBT2011 transforms according to above detected result, its converted product is the pre-sapogenin of potato.
The measuring method of Determination of Diosgenin (RPLC RP-HpLC): Agilent1100 highly effective liquid phase chromatographic system; Chromatographic column: ZorbaxC18 (150mm * 4.6mmi.d.), moving phase: methanol/water=90/10 (v/v), detecting wavelength: 210nm, flow velocity: 1mL/min, column temperature are normal temperature.
Embodiment 2
Basic identical with embodiment 1, difference is:
Add 600g yellow ginger dry powder and 3000mL tap water in the 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
20.03g Deng becoming to be distributed into the conversion slurries, 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 sterilization, at 40 ℃, 400rpm, 3.5 l/min transform 72hrs under the culture condition of control pH 6.0.Transform slurries after filtration, drying finally obtains hydrolyzate dry weight 198.7g, by interpolation yellow ginger dry powder dry weight 33.1%.Hydrolyzate is with 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid, and adds industrial gac depigmentation, obtains diosgenin finished product 16.5g through periodic crystallisation, and the 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 dried yellow ginger powder of 250g and 3000mL water in the 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
20.03g Deng becoming to be distributed into the conversion slurries, 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 sterilization, in 40 ℃, 300rpm, 2.5 l/min transform 56hrs under the culture condition of control pH 6.2.Cultivate slurries after filtration, drying obtains hydrolyzate dry weight 70.5g, by interpolation yellow ginger dry powder dry weight 28.2%.Hydrolyzate is with 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid, and adds industrial gac depigmentation, obtains 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.The thin-layer chromatography result shows that the transformation efficiency of total dioscin is 100%.
Described yeast extract is take 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%, the diosgenin yield is 2.7～3.05%.This processing method can make total dioscin approach and change into diosgenin fully, treatment capacity reaches industrialized level, and the waste liquid amount that produces is about 1/3 of present industrialization status, and does not contain sugar, pH can reduce environmental pollution to greatest extent close to neutrality.
The part that the present embodiment is not described in detail and english abbreviation belong to the common practise of the industry, can search on the net, here not narration one by one.
Claims (6)
1. produced the method for diosgenin through microbial fermentation by Rhizome of Peltate Yam-yellow ginger for one kind, 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 urea into 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 the conversion slurries, the access volume be about tap water volume 5% bacillus (
Baccilus) strain X BT2011 seed liquor, 40 ℃ 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, and drying obtains hydrolyzate, hydrolyzate obtains the diosgenin finished product with 120 long-pending ﹟ gasoline extraction 12-16hrs of monoploid through periodic crystallisation; Described rpm is rev/min, and hrs is hour; Described bacillus (
Baccilus) preserving number of strain X BT2011: CGMCCNo.6301.
2. a kind of method of diosgenin of being produced through microbial transformation by Rhizome of Peltate Yam-yellow ginger according to claim 1, is characterized in that: in conversion process, get the 5ml conversion fluid every 6hrs and detect diosgenin conversion situation from fermentor tank; The water-saturated n-butanol reagent that adds 2 times of liquid sample volumes in the liquid sample, high speed centrifugation 5min after ultrasonic echography 20min, get supernatant liquor to the crucible in 100 ℃ of oven dry, the methanol solution of liquid sample volume again doubles in the crucible, through repeatedly filtering after dissolving, getting appropriate filtrate point sample on thin layer chromatography board, is that the chloroform/methanol/water developping agent of 7: 3: 0.5 is launched with volume ratio, spray 10ml mass percent concentration 10% H
2SO
4Solution heating colour developing with the contrast of the pre-sapogenin mark of potato product, is observed hydrolysis degree and the diosgenin transforming degree of total dioscin; Cultivate slurries and filter through industrial filter cloth, obtain hydrolyzate in 85 ℃ of oven dry, the 120 ﹟ gasoline extraction 12-16hrs that hydrolyzate amasss with about monoploid, and add industrial gac depigmentation, obtain the diosgenin finished product through periodic crystallisation.
3. according to claim 1 and 2 a kind of by the method for Rhizome of Peltate Yam-yellow ginger through microbial transformation production diosgenin, it is characterized in that: gained hydrolyzate dry weight in the method, by being added yellow ginger dry powder dry weight 28～33%, the diosgenin yield is 2.7～3.05%.
4. according to claim 1 a kind of by the method for Rhizome of Peltate Yam-yellow ginger through microbial transformation production diosgenin, it is characterized in that: described inorganic salt are N
aCl, MgSO
4, k
2HPO
4, FeSO
4And CaCl
2Mixture.
5. according to claim 1 or 4 is described a kind of by the methods of Rhizome of Peltate Yam-yellow ginger through microbial transformation production diosgenin, and it is characterized in that: described inorganic salt are to be divided into 0.1% N by the quality volume percent
aCl, 0.05%MgSO
4, 0.05%k
2HPO
4, 0.005%FeSO
4And 0.001%CaCl
2Form.
6. according to claim 1 a kind of by the method for Rhizome of Peltate Yam-yellow ginger through microbial transformation production diosgenin, it is characterized in that: being prepared as of described seed liquor got the 5ml cell density 10
8～10
9/ ml's
Bacillus aryabhattaiThe XBT2011 suspension inoculation at 40 ℃, is cultivated 14hrs and is made in the 150ml seed culture fluid under the shake-flask culture condition of 200rpm; Wherein said seed culture fluid is comprised 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%.
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