CN110093391A - The method that microorganism conversion prepares caffeine - Google Patents
The method that microorganism conversion prepares caffeine Download PDFInfo
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- CN110093391A CN110093391A CN201810085392.4A CN201810085392A CN110093391A CN 110093391 A CN110093391 A CN 110093391A CN 201810085392 A CN201810085392 A CN 201810085392A CN 110093391 A CN110093391 A CN 110093391A
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- caffeine
- microorganism conversion
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- eurotium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
Abstract
The present invention provides a kind of methods that microorganism conversion prepares caffeine, using decaffeinated urban afforestation waste and agricultural waste material as substrate in the reaction system of microorganism conversion, with E.intermedius (Eurotium intermedium, CGMCC 3.4318), thank to a watt bulk bacteria (Eurotium chevalieri, CGMCC 3.6492), coronoid process dissipate capsule bacterium (Eurotium cristatum, CGMCC 3.0448), E.amstelodami (Eurotium amstelodami, CGMCC 3.6518), wax leaf bulk bacteria (Eurotium herbarioum, CGMCC 3.6496) one or more single mixed bacterias in as microorganism conversion strain, cultivate 1~15 day at a temperature of being 15 DEG C~45 DEG C by conversion condition.The present invention provides the method that microorganism conversion prepares caffeine, solves and prepare caffeine process solvent be added and the easy residual harmful substance of catalyst in the prior art, and the technical problem expensive using equipment, technique is cumbersome, at high cost.
Description
Technical field
The present invention relates to biosynthesis technology fields, particularly, are related to a kind of method that microorganism conversion prepares caffeine.
Background technique
Caffeine is a kind of astragalus alkaloid compound, is a kind of central nervous excitation agent, can temporarily drive away sleepiness
And regain one's vigor, clinically for treating neurasthenia and stupor recovery.There are coffee, tea, soft drink and the energy of caffeine ingredient
Amount beverage is in very great demand, and therefore, caffeine is also the psychotropic substances being most commonly used in the world.In North America, 90% adult
Caffeine is all used daily.
There are mainly three types of the modes for obtaining caffeine at present, the first is to be led to by chemical synthesis by monoxone or cyanoacetic acid
Cross complicated chemical reaction commercial synthesis caffeine；Second is by way of solvent extraction or supercritical extract from Gao Chengben
The natural plants such as tealeaves or coffee bean rich in caffeine in extract caffeine；The third is by caffeine at 100 DEG C
The characteristic that distillation is easy when above extracts coffee from the high-cost natural plants rich in caffeine such as tealeaves or coffee bean
Cause.The mode of above-mentioned three kinds of acquisitions caffeine has fatal defect, largely uses harmful substance as reaction in chemical synthesis
Solvent and catalyst have certain residual in final product and then potential hazard constituted to human body, while to ecological ring
Border causes irreversible destruction；Latter two mode is in addition to using a large amount of organic reagents to constitute harm, technique to environment and human body
Outside the obvious disadvantages such as the cumbersome low extent of damage of extraction efficiency is high, equipment valuableness operating cost is huge, it is often more important that this two
The maximum of kind of mode is on condition that must be premised on the natural plants (coffee bean or tealeaves) rich in caffeine, and this type is natural
Plant (coffee bean or tealeaves) cost is higher, for caffeine industrial volume production this can mean that huge cost input,
This caffeine product market price for just directly resulting in current plant source is up to thousands of first per kilograms.
Currently, having no a kind of preparation method of efficient, green, convenient, inexpensive synthetic caffeine in the market.
Summary of the invention
The present invention provides a kind of methods that microorganism conversion prepares caffeine, solve and prepare caffeine in the prior art
The solvent and catalyst that process is added are easy residual harmful substance, and the skill expensive using equipment, technique is cumbersome, at high cost
Art problem.
To achieve the above object, the invention proposes a kind of method that microorganism conversion prepares caffeine, microorganism conversions
Reaction system in using decaffeinated urban afforestation waste and agricultural waste material as substrate, with E.intermedius
(Eurotium intermedium, CGMCC 3.4318), a watt bulk bacteria (Eurotium chevalieri, CGMCC are thanked
3.6492), coronoid process dissipate capsule bacterium (Eurotium cristatum, CGMCC 3.0448), E.amstelodami (Eurotium
Amstelodami, CGMCC 3.6518), one in wax leaf bulk bacteria (Eurotium herbarioum, CGMCC 3.6496)
The strain of kind or a variety of strains as microorganism conversion, conversion condition are cultivated 1~15 day at a temperature of being 15 DEG C~45 DEG C.
Preferably, the tunning be strain used product after liquid fermentation or solid fermentation evaporated, depressurize it is dry
The medicinal extract obtained after dry processing.
Preferably, at least 2 times activation processing are carried out before the strain fermentation, are covered with bacterium colony in culture to culture medium, are taken out
Strain is suspended in sterile water after activation, then is mixed shaking after suspension constant volume, and uniform spore suspension, the spore is made
The spore concentration of suspension is 5.0~6.0 × 108CFU/ml, spore inoculating amount are 1%~20%.
Preferably, the solid fermentation includes: that spore suspension Direct Uniform is sprayed at urban afforestation waste and farming
Object waste surface, by whole fermentation after waste itself is cultivated 1~15 day in sterile culture case as the carbon source of fermentation
For product through extraction with aqueous solution, suction filtration, products therefrom is the product of solid fermentation.
Preferably, the liquid fermentation includes: by decaffeinated urban afforestation waste and agricultural waste material system
At powder, sterile water being added, decocted, filtered, gained filtrate is substrate extracting solution, it is added 0 in the substrate extracting solution~
Carbon source of 20% glucose as fermentation, adjustment pH are 3.0~8.0, and spore suspension is added in substrate extracting solution and is shaken
Culture obtains the product that upper layer suspension is liquid fermentation after centrifugation.
Preferably, it is 1/20~2/3 shaking flask amount, shake culture that the spore suspension, which is added to liquid amount in substrate extracting solution,
Revolving speed be 0~180r/min.
Preferably, the substrate of the reaction system of the microorganism conversion is selected from ginkgo leaf, mulberry leaf, capsicum leaf, Poplar leaves, Chinese scholartree
One of leaf, Folium Pterocaryae.
Preferably, microorganism conversion product is through Amberlyst process extracting caffeine, using 35% ethyl alcohol as eluant, eluent.
Caffeine made from the method for caffeine, purity are prepared as mentioned microorganism conversion the invention also provides a kind of
Higher than 98%.
The utility model has the advantages that
The embodiment of the invention provides a kind of methods that microorganism conversion prepares caffeine, green with decaffeinated city
Changing waste and agricultural waste material is raw materials for production, prepares caffeine by microorganism conversion means, utilizes to greatest extent
Urban afforestation and agricultural wastes, promote the cycling and reutilization of its high added value, efficient convenient, the green ring of the method for the present invention
It protects, at low cost, product purity is high, market prospects are huge.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, With reference to embodiment
The present invention is described in further detail.
Strain of the present invention is purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center.Institute
It is commercial goods with instrument, reagent.
The method that microorganism conversion of the present invention prepares caffeine, specifically includes:
Dry raw material powder is taken, sterile water is added, is decocted, filters, filtrate is obtained, after residue plus aseptic water washing 2 times
Merge as in filtrate, constant volume obtains bioconversion substrate extracting solution.
Substrate extracting solution is transferred in the shaking flask of 2L capacity, carbon source of the glucose as fermentation is added, then adjust pH；It will
The substrate extracting solution prepared carries out 121 DEG C autoclave sterilization 20 minutes；It is prepared in this approach for microorganism conversion
Fluid nutrient medium, it is spare.
Tunning is prepared by liquid fermentation, comprising: before strain is inoculated into fluid nutrient medium, is stored in 4 DEG C of rings
Bacterial strain under border should be activated 2 times in plating medium, and activation is in 30 DEG C of constant incubators (DH-360 type electric heating every time
Constant incubator) middle culture 4 days.Bacterium colony is covered on plating medium after 4 days, under aseptic technique, to plate culture
Sterile water is poured into base, and is scraped strain with oese, is suspended in it in this sterile water, then suspension is poured into and fills glass
In the sterile water of glass pearl, constant volume.Again with 150 revs/min, concussion is mixed 20 minutes, and uniform spore suspension is made.Measure spore
Quantity, spore concentration should be 5.0~6.0 × 108CFU/ml, spore inoculating amount are 1%~20%.Spore suspension is poured into system again
In the fluid nutrient medium got ready, shaken cultivation under the conditions of certain revolution and temperature.Centrifugation separates bacterium solution, and bacterium falls to down
Layer, obtains high fermentation liquid, rotary evaporation is dried under reduced pressure to obtain medicinal extract, as tunning.
Tunning is prepared by solid fermentation, comprising: by spore suspension obtained in aforesaid liquid fermentation preparation flow
Direct Uniform is sprayed on substrate blade, is cultivated in sterile culture case after a certain period of time, and tunning is flowed back with aqueous solution
It extracts, rotary evaporation is dried under reduced pressure to obtain medicinal extract, as tunning.
Preferably, sugaring amount when bioconversion is 0~20%；Solution acid alkalinity (pH value) when bioconversion is 3.0
~8.0；Cultivation temperature when bioconversion is 15~45 DEG C；The bacterium amount that connects when bioconversion is 1~20%；When bioconversion
1/20 shaking flask amount of liquid amount is to 2/3 shaking flask amount；Shaking speed when bioconversion is 0~180r/min；Training when bioconversion
Supporting number of days is 1~15 day.
Under the conditions of above-mentioned preferred, by microorganism conversion products therefrom (above-mentioned tunning), by applying macropore
Resin filtering, and separated using 35% ethyl alcohol as eluent, it is 234.7~237.4 DEG C in combination with melting point of caffeine
Physical characteristic, purifying obtain the caffeine that purity is greater than 98%.
Embodiment 1
Using ginkgo leaf as substrate, coronoid process dissipate capsule bacterium is as microorganism conversion strain.
It takes 300mL substrate extracting solution to be sub-packed in 3 250mL conical flasks, is separately added into 6g glucose, adjust pH to 5.5,
Autoclave sterilization (121 DEG C of 20min).
Coronoid process dissipate capsule bacterium is cultivated on PDA plate culture medium, plate covers with bacterium colony after 4d, under aseptic technique,
Sterile water about 10mL is poured into, yellow cleistothecium is scraped with oese, is suspended in sterile water, then suspension is poured into and fills glass
In the sterile water of glass pearl, 150r/min, concussion mixes 20min, and uniform spore suspension is made.Spore quantity is measured, spore is dense
Degree is 6.0 × 108CFU/mL, spore inoculating amount are 20%.
Every bottle plus 5mL bacterium solution, shake culture 0,7,15 day under the conditions of 120r/min, 30 DEG C.Finally by using macropore
Resin filtering, and using 35% ethyl alcohol as the isolated caffeine crude extract of eluent, caffeine crude extract is placed in steaming
It sends out in ware, and admixes 20g calcium oxide, in evaporating water on electric jacket, recycling melting point of caffeine is 234.7~237.4 DEG C
Physical characteristic, heating make its distillation, and purification obtains caffeine, detected by HPLC liquid phase, and purity is greater than 98%.
Embodiment 2
Using mulberry leaf as substrate, to thank to watt bulk bacteria as microorganism conversion strain.
It takes 500mL bioconversion substrate extracting solution to be sub-packed in 5 250mL conical flasks, is separately added into 0,2,4,8, the Portugal 20g
Grape sugar, adjusts pH to 5.5, autoclave sterilization (121 DEG C of 20min).
Culture thanks to a watt bulk bacteria on PDA plate culture medium, and plate covers with bacterium colony after 10d, under aseptic technique,
Sterile water about 10mL is poured into, cleistothecium is scraped with oese, is suspended in sterile water, then suspension is poured into and fills bead
Sterile water in, 150r/min, concussion mix 20min, uniform spore suspension is made.Spore quantity is measured, spore concentration is
5.0×108CFU/mL, spore inoculating amount are 5%.
Every bottle plus 5mL bacterium solution, shake culture 4 days under the conditions of 120r/min, 30 DEG C.Finally by using macroreticular resin mistake
Filter, and using 35% ethyl alcohol as the isolated caffeine crude extract of eluent, caffeine crude extract is placed in evaporating dish,
And 20g calcium oxide is admixed, in evaporating water on electric jacket, recycle the physics that melting point of caffeine is 234.7~237.4 DEG C special
Property, heating makes its distillation, and purification obtains caffeine, is detected by HPLC liquid phase, and purity is greater than 98%.
Embodiment 3
Using Poplar leaves as substrate, using E.intermedius as microorganism conversion strain.
It takes 500mL bioconversion substrate extracting solution to be sub-packed in 5 250mL conical flasks, is separately added into 6g glucose, adjust
PH to 3,5,5.5,6,8, autoclave sterilization (121 DEG C of 20min).
E.intermedius is cultivated on PDA plate culture medium, plate covers with bacterium colony after 4d, under aseptic technique,
Sterile water about 10mL is poured into, its cleistothecium is scraped with oese, is suspended in sterile water, then suspension is poured into and fills glass
In the sterile water of pearl, 150r/min, concussion mixes 20min, and uniform spore suspension is made.Measure spore quantity, spore concentration
It is 5.5 × 108CFU/mL, spore inoculating amount are 12%.
Every bottle plus 5mL bacterium solution, shake culture 11 days under the conditions of 120r/min, 30 DEG C.Finally by using macroreticular resin
Filtering, and using 35% ethyl alcohol as the isolated caffeine crude extract of eluent, caffeine crude extract is placed in evaporating dish
In, and 20g calcium oxide is admixed, in evaporating water on electric jacket, recycling melting point of caffeine is 234.7~237.4 DEG C of physics
Characteristic, heating make its distillation, and purification obtains caffeine, detected by HPLC liquid phase, and purity is greater than 98%.
Embodiment 4
Using Folium sophorae as substrate, using E.amstelodami as microorganism conversion strain.
E.amstelodami is cultivated on PDA plate culture medium, plate covers with bacterium colony after 4d, in sterile working item
Under part, sterile water about 10mL is poured into, its cleistothecium is scraped with oese, is suspended in sterile water, then suspension is poured into Sheng
Have in the sterile water of bead, 150r/min, concussion mixes 20min, and uniform spore suspension is made.Measure spore quantity, spore
Sub- concentration is 5.5 × 108CFU/mL, spore inoculating amount are 10%.
Spore suspension is sprayed on the blade of Folium sophorae, after being cultivated 10 days in sterile culture case, tunning is used
Aqueous solution refluxing extraction, rotary evaporation are dried under reduced pressure to obtain medicinal extract.It is filtered using macroreticular resin, and using 35% ethyl alcohol as elution
Agent elutes isolated caffeine crude extract, caffeine crude extract is placed in evaporating dish, and admix 20g calcium oxide, in electric heating
Evaporating water is put on, recycling melting point of caffeine is 234.7~237.4 DEG C of physical characteristic, and heating makes its distillation, and purification obtains
Caffeine is detected by HPLC liquid phase, and purity is greater than 98%.
The embodiment of the present invention has been described in detail above, specific case used herein to the principle of the present invention and
Embodiment is expounded, and the above description of the embodiment is only used to help understand the method for the present invention and its core ideas；
At the same time, for those skilled in the art can in specific embodiments and applications according to the thought of the present invention
There is change place, in conclusion the contents of this specification are not to be construed as limiting the invention.
Claims (9)
1. the method that microorganism conversion prepares caffeine, which is characterized in that be free of coffee in the reaction system of microorganism conversion
The urban afforestation waste and agricultural waste material of cause as substrate, with E.intermedius (Eurotium intermedium,
CGMCC 3.4318), thank a watt bulk bacteria (Eurotium chevalieri, CGMCC 3.6492), coronoid process dissipate capsule bacterium
(Eurotium cristatum, CGMCC 3.0448), E.amstelodami (Eurotium amstelodami, CGMCC
3.6518), one of wax leaf bulk bacteria (Eurotium herbarioum, CGMCC 3.6496) or a variety of strains are as micro-
Bioconversion strain, conversion condition are cultivated 1~15 day at a temperature of being 15 DEG C~45 DEG C.
2. the method that microorganism conversion prepares caffeine according to claim 1, which is characterized in that the tunning is institute
With strain, product is evaporated, is dried under reduced pressure the medicinal extract obtained after handling after liquid fermentation or solid fermentation.
3. the method that microorganism conversion prepares caffeine according to claim 2, which is characterized in that the strain fermentation advances
It goes at least 2 times and is activated, bacterium colony is covered in culture to culture medium, strain is suspended in sterile water after taking out activation, then will be hanged
It shaking and mixes after supernatant liquid constant volume, be made uniform spore suspension, the spore concentration of the spore suspension is 5.0~6.0 ×
108CFU/ml, spore inoculating amount are 1%~20%.
4. the method that microorganism conversion prepares caffeine according to claim 2, which is characterized in that the solid fermentation packet
Include: spore suspension Direct Uniform be sprayed at urban afforestation waste and agricultural waste material surface, using waste itself as
The carbon source of fermentation is cultivated whole tunning after 1-15 days specified times through extraction with aqueous solution in sterile culture case, is filtered,
Products therefrom is the product of solid fermentation.
5. the method that microorganism conversion prepares caffeine according to claim 2, which is characterized in that the liquid fermentation packet
It includes: powder is made in decaffeinated urban afforestation waste and agricultural waste material, sterile water is added, decocted, taken out
Filter, gained filtrate are substrate extracting solution, and carbon source of the glucose of addition 0~20% as fermentation, is adjusted in the substrate extracting solution
Whole pH is 3.0~8.0, and spore suspension is added to shake culture in substrate extracting solution, and it is liquid hair that upper layer suspension is obtained after centrifugation
The product of ferment.
6. the method that microorganism conversion prepares caffeine according to claim 5, which is characterized in that the spore suspension is added
Into substrate extracting solution, liquid amount is 1/20~2/3 shaking flask amount, and the revolving speed of shake culture is 0~180r/min.
7. the method that microorganism conversion prepares caffeine according to claim 1, which is characterized in that the microorganism conversion
The substrate of reaction system is selected from one of ginkgo leaf, mulberry leaf, capsicum leaf, Poplar leaves, Folium sophorae, Folium Pterocaryae.
8. the method that microorganism conversion prepares caffeine according to claim 1, which is characterized in that microorganism conversion product warp
Amberlyst process extracting caffeine, using 35% ethyl alcohol as eluant, eluent.
9. a kind of prepare caffeine made from the method for caffeine as any microorganism conversion of claim 1 to 9, purity is high
In 98%.
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| CN201810085392.4A CN110093391A (en) | 2018-01-29 | 2018-01-29 | The method that microorganism conversion prepares caffeine |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651424A (en) * | 2004-11-25 | 2005-08-10 | 三达膜科技（厦门）有限公司 | Production method of high purity tea polyphenol and caffeine |
| CN104893984A (en) * | 2015-04-07 | 2015-09-09 | 安徽农业大学 | Eurotium cristatum strain |
-
2018
- 2018-01-29 CN CN201810085392.4A patent/CN110093391A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1651424A (en) * | 2004-11-25 | 2005-08-10 | 三达膜科技（厦门）有限公司 | Production method of high purity tea polyphenol and caffeine |
| CN104893984A (en) * | 2015-04-07 | 2015-09-09 | 安徽农业大学 | Eurotium cristatum strain |
Non-Patent Citations (4)
| Title |
|---|
| 张东方等: "《中药现代分离技术》", 31 May 2006 * |
| 林树钱: "《中国药用菌生产与产品开发》", 30 November 2000, 中国农业出版社 * |
| 詹欣: "银杏叶发酵产物的生物活性检测与分离及其发酵条件优化", 《中国优秀硕士学位论文全文数据库》 * |
| 陈勇强等: "茯砖茶中几种常见散囊菌与曲霉的对比研究", 《福建分析测试》 * |
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