CN101260414B - Technique for preparing ribitol by fermentation method - Google Patents
Technique for preparing ribitol by fermentation method Download PDFInfo
- Publication number
- CN101260414B CN101260414B CN2008100235961A CN200810023596A CN101260414B CN 101260414 B CN101260414 B CN 101260414B CN 2008100235961 A CN2008100235961 A CN 2008100235961A CN 200810023596 A CN200810023596 A CN 200810023596A CN 101260414 B CN101260414 B CN 101260414B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- ribitol
- glucose
- controlled
- dissolved oxygen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a preparing technology for preparing ribitol by zymotechnics, which uses glucose as raw material and Trichosporonoides oedocephalis as original strain, and prepares according to the steps of strain preparation, seed cultivation and fermentation. The invention is characterized in that seed solution and fermentation medium are inoculated in a fermentation cylinder in accordance with the volume ratio between 0.1 and 0.01, and are agitated for fermentation cultivation; the fermentation cultivation is performed under the technological condition of the cylinder pressure between 0 and 1MPa, the temperature between 25 and 40 DEG C, the dissolved oxygen between 5 and 95 percent and the pH between 4 and 7; the time for the process of fermentation cultivation is between 80 to160h; the agitation for fermentation process is sectional agitation; and the dissolved oxygen is sectionally controlled through sectionally controlling the speed of agitation. Sectional feeding and purification are implemented. The preparing technology for preparing ribitol by zymotechnics has the characteristics of simple processes, high yield, small material consumption, low content in glyceroland other side products, steady product quality, low preparation cost, and no discharge of three wastes, etc.
Description
Technical field
The present invention relates to a kind of preparation technology of ribitol, be specifically related to a kind of preparation technology who adopts fermentation method to prepare ribitol.Belong to technical field of biochemical industry.
Background technology
Ribitol is the important starting raw material of preparation high value non-natural sugar, it can through the microbial enzyme reaction directly transform make L-ribose or be prepared into the L-ribulose by the microorganism dehydrogenation after re-isomerization become L-ribose.And non-natural in recent years L-type sugar and their modification nucleoside analog have begun to enjoy the concern of Many researchers because of its potential biologic activity and hypotoxicity, be used to prepare the sweet analogue of the nuclear with anti HIV-1 virus effect as rare L-pentose (L-ribose, L-wood sugar etc.), the sweet analogue of the nuclear of present a plurality of L-ribose be in the II phase or III clinical.Can predict, along with the development as the novel antiviral medicine such as the sweet analogue of nuclear of L-ribose etc., ribitol also will have vast market prospect.
At present, the preparation of ribitol normally adopts chemical process that the reduction of D-ribose is obtained.Yet D-ribose itself promptly can be directly used in disease treatment as medicine in medicine industry, can be used as the synthetic other drug of precursor substance again, thereby D-ribose price is higher relatively.On the other hand, the hydrogenation of D-ribose needs High Temperature High Pressure in the chemical preparation process of ribitol, and organic molten coal has brought trouble and both expensive for again aftertreatment and environmental protection.Therefore, for adapting to many-sided demands such as market, friendly process, the development biotechnology directly becomes ribitol to have important economy and social effect the renewable resources such as the conversion of glucose of cheapness.
1998 Japanese Patent (JP10210994) announced the method that the cheap sugar of t bacteria richosporonoides shake flask fermentation is produced ribitol.Its technology characteristics is to make ribitol with the glucose one-step fermentation.The wherein inevitable by products such as a large amount of erythritols and glycerine that produce.1999 Japanese Patent (JP11137285) further announced with glucose to be raw material, adopt the method for t bacteria richosporonoidesoedocephalis ATCC 16958 fermentative production ribitol, its main technique feature is to utilize high concentration glucose fermentative production ribitol, product separates storng-acid cation exchange resins such as adopting aluminium type, manganese ribitol is separated, by products such as the same inevitably generation of this technology characteristics glycerine, separation costs is higher relatively simultaneously.The same year, Japanese Patent (JP11004699) has been announced the separation of ribitol and has been reclaimed technology, after its technology characteristics is somatic cells removed, in fermentation clear liquid, add the white lime after-filtration, after add small molecular alcohol such as methyl alcohol in the filtrate with the ribitol precipitation, this technological process produces great amount of carbon dioxide, sepn process alcohol large usage quantity, it is low that product purity is separated the product that obtains than ion exchange resin, is unfavorable for suitability for industrialized production.In addition, 1999 Japanese Patent (JP11215981) also announced the substratum of producing ribitol, main technique is characterised in that utilizes flesh of fish extract, soyflour, defatted soyflour or cottonseed protein etc. to produce ribitol as nitrogenous source.
Up to the present, except that above-mentioned patent, do not see as yet that both at home and abroad other is arranged is raw material with glucose, adopt fermentation method to prepare ribitol preparation technology's report.
Summary of the invention
It is fairly simple that the present invention aims to provide a kind of preparation technology, the finished product steady quality, and preparation cost is lower, and the employing fermentation method of basic three-waste free discharge prepares the preparation technology of ribitol.
The present invention realizes that the overall conception of its purpose is, with reproducible cheap glucose is raw material, select for use height to ooze yeast bulb three type spore bacterium (Tricnosporonoides oedocephaIis) and be starting strain, according to strain preparation, seed culture and three steps of fermentation are carried out fermentation culture successively.And in its fermentation culture process, take segmentation to stir measure, control the dissolved oxygen amount of its fermenting process; Take segmentation feed supplement measure simultaneously,, build the cultivation that meets the bacterial classification microorganism, the condition of breeding and help the fermentative preparation ribitol, the concentration of ribitol in the combined regulating fermented liquid and byproduct glycerine by segmentation stirring and segmentation feed supplement; And, produce high-quality, high purity white crystals ribitol by the purification of fermented liquid.
Because above-mentioned conception, the present invention realizes that the technical scheme of its purpose is:
A kind of preparation technology who adopts fermentation method to prepare ribitol is a raw material with glucose, and oozing yeast bulb three type spore bacterium with height is starting strain, and successively according to strain preparation, seed culture and three steps of fermentation are carried out, and its innovative point is:
A, described fermentation step are seed liquor that step 2 is prepared with the fermention medium that contains glucose by 1～10% volume ratio, be inoculated in the sterilized fermentor tank that described fermention medium is housed, implement to stir fermentation culture, produce fermented liquid;
B, described fermentation culture are to be 0～1Mpa at tank pressure, and temperature is 25～40 ℃, and dissolved oxygen is 5～95%, and PH carries out under 4～7 the processing condition; The time of fermentation culture process is 80～160h, to the residual sugar amount≤0.3% o'clock fermentation ends of its glucose;
The stirring of c, described fermenting process is that segmentation is stirred; The mixing speed of preceding 30h is controlled in 200～600rpm scope, and dissolved oxygen is controlled in 65～95% scopes; Mixing speed behind the 30h is controlled in 100～300rpm scope, and dissolved oxygen is controlled in 5～65% scopes.
The strain preparation that technique scheme is related can adopt common mode, is about to its described inoculation inclined-plane, under 25～35 ℃ of constant temperatures, behind cultivation 90～100h, preserves stand-by under 1～4 ℃ of temperature condition.
And described seed culture can adopt common mode equally, is about to prepared inclined-plane, is inoculated in the shake-flask seed substratum; Culture temperature is controlled in 25～35 ℃ of scopes, and shaking speed is controlled in 100～250rpm scope, and incubation time is 45～50h.And the weight content of the component of its described substratum and each component, what the present invention recommended is: glucose 150～250g/L, yeast soak powder 5～10g/L, and MgSO4.7H2O is 0.2～0.8g/L, and NaCl is 0.2～0.8g/L, prepares with pure water.
And the described component of the fermention medium in the fermentor tank and the weight content of each component of being contained in, the fermention medium that contains glucose is basic identical with having, what the present invention recommended is: glucose is 50～250g/L, it is 15～25g/L that yeast soaks powder, MgSO4.7H2O is 0.2～0.8g/L, NaCl is 0.2～0.8g/L, and defoamer (bubble enemy) is 2～5mL/L, prepares with pure water.And the weight content of described glucose can be implemented the segmentation regulation and control in whole fermentation process.
The present invention implements according to technique scheme, and as seen its raw material route and technical process are all fairly simple, and the fermentation culture process control is easy, and energy consumption is lower, and preparation cost is lower, three-waste free discharge.Thereby realized purpose of the present invention.
The present invention also advocates, the glucose in the described fermention medium, and segmentation adds in the described fermentor tank in the fermentation culture process; In earlier fermentation 0～30h time range, the control glucose concn is in 5～15% scopes; Behind 30h, add glucose and KH
2PO
4, and control glucose concn in 5～25% scopes, described KH
2PO
4Add-on be controlled in 0.1～1.5g/L scope.Why will implement to stir the segmentation feed supplement of reply mutually with segmentation, its purpose is to build the condition that is fit to the bacterial classification microorganism culturing more, breeds and help the fermentative preparation ribitol, with the consumption of the used fermention medium material of effective reduction, reduces preparation cost; And thus can be by segmentation stirring and segmentation feed supplement, the concentration (content) of ribitol in the combined regulating fermented liquid and byproduct glycerine.
The present invention also advocates, behind fermentation step, also comprises purification step; Described purification step is the fermented liquid that will produce by step 3, removes thalline through centrifugation, activated carbon decolorizing, and underpressure distillation concentrates, chromatographic separation resin chromatography, chromatographic solution concentrates and the organic solvent recrystallization, and gets the ribitol product of high purity white crystals.
The concrete steps of the described purification that the present invention recommends are: with the fermented liquid of glucose residual sugar amount≤0.3%, remove thalline through the 5000g/10min centrifugation; Again through activated carbon decolorizing; It is 40～90% that underpressure distillation is concentrated into ribitol concentration; Then through chromatographic separation resin chromatography; Collection contains the ribitol elutriant, again through the organic solvent ethyl alcohol recrystallization, gets the high purity white crystals ribitol goods of content 〉=99% after concentrating.And the elutriant of described chromatographic separation process is a pure water; The separator column aspect ratio is 10～40, and the best is 15～35; Elution flow rate is 1～25mL/min, and the best is 2～15mL/min.
The described chromatographic separation resin that the present invention advocated is a Ca type storng-acid cation exchange resin.And the present invention recommended is that Britain produces PUROLITE PCR642 calcium type ion exchange resin, or homemade 001 * 7 type and D001 type strongly-acid sun sub-exchange resin, through the Cacl of 0.5mol/L
2The Ca type storng-acid cation exchange resin of transition.
After technique scheme is implemented, the present invention stirs dissolved oxygen amount and the segmentation feed supplement mode of controlling fermenting process by segmentation, and combined regulating ribitol production concentration reduces by-product glycerin content, and the technology that is had is simple, ribitol productive rate height, it is few to consume raw material, and by-products contents such as glycerine are low, constant product quality, characteristics such as the low and three-waste free discharge of preparation cost are conspicuous.Compared with the prior art, the present invention has outstanding substantive distinguishing features and marked improvement.
Description of drawings
Fig. 1 is the process flow sheet of a kind of embodiment of the present invention;
Fig. 2 is the process flow sheet of the another kind of embodiment of the present invention;
Fig. 3 is a purification step process flow sheet of the present invention;
Fig. 4 is the ribitol fermentation liquor liquid chromatogram of a kind of embodiment of the present invention;
Fig. 5 is the ribitol fermentation liquor liquid chromatogram of another kind of embodiment segmentation control dissolved oxygen of the present invention and feed supplement;
Fig. 6 is the infrared spectrum of goods ribitol product of the present invention.
Embodiment
Below description by embodiment, the invention will be further described.
Embodiment 1: as shown in Figure 1.Its processing step is:
(1) bacterial strain is selected for use.Select for use height to ooze yeast bulb three type spore bacterium (Trichosporonoidesoedocephalis ATCC 16958)
(2) medium preparation.
(3) strain preparation.With described inoculation inclined-plane, after 30 ℃ ± 1 ℃ temperature is cultivated 4 days, place 4 ℃ of refrigerators stand-by.
(4) seed culture.
The bacterial classification of step 3 slant culture after 4 days inserted in the seed culture fluid.Seed culture adopts aerobic to cultivate, and uses the 500mL triangle to shake bottle, and liquid amount is 100mL, and culture temperature is 30 ℃, and shaking speed is 170rpm, and incubation time is 45h.
(5) fermentation culture.
A, fermentation culture adopt the 50L fermentor tank, and liquid amount is 30L, and tank pressure is 0.5MPa, and culture temperature is 30 ℃, and pH is 5.0;
B, fermentation mode
The seed culture fluid of step 4 preparation is inserted in the fermention medium with 5% volume ratio, and the air flow of the whole process of fermenting is controlled to be 1vvm, and dissolved oxygen is controlled at about 65%, and the rotating speed that segmentation is stirred is 350rpm.
The result is as follows for c, fermentation
Fermentation was carried out 120 hours altogether, and glucose is dense to be transformed fully, and high performance liquid chromatography area normalization fractional analysis as shown in Figure 4 contains glycerine 21g/L in the fermented liquid, erythritol 8.5g/L, and ribitol 45g/L, ribitol production process inversion rate of glucose is 22.5%.
Embodiment 2: as shown in Figure 1.Its processing step is:
(1) bacterial classification is selected for use.With embodiment 1.
(2) medium preparation.With embodiment 1.
(3) strain preparation.With embodiment 1.
(4) seed culture.With implementing 1.
(5) fermentation culture.
A, fermentation culture adopt the 50L fermentor tank, and liquid amount is 30L, and tank pressure is 0.5MPa, and culture temperature is 30 ℃, and pH is 5.0;
B, fermentation mode
The seed culture fluid of step 4 preparation is inserted in the fermention medium with 5% volume ratio, the air flow of the whole process of fermenting is controlled to be 1vvm, the preceding 30 hours mixing speed that ferment are 350rpm, dissolved oxygen is controlled in 65～95% scopes, after 30 hours mixing speed is reduced to 100～200rpm, make dissolved oxygen be controlled at 5%～30%, promptly adopt segmentation of the present invention to stir fermentation method.
(6) fermentation was carried out 120 hours altogether, and glucose transforms fully, contains glycerine 15g/L in the efficient liquid phase chromatographic analysis ferment liquid, erythritol 7.6g/L, and ribitol 65g/L, ribitol production process inversion rate of glucose is 32.5%.
Embodiment 3: as shown in Figure 1.Its processing step is:
(1) bacterial classification is selected for use.With embodiment 1.
(2) medium preparation.With embodiment 1.
(3) strain preparation.With embodiment 1.
(4) seed culture.With implementing 1.
(5) fermentation culture.
A, seed culture fluid is inserted in the fermention medium with 5% volume ratio, air flow is controlled to be 1vvm in the whole process of fermentation, the preceding 30 hours mixing speed that ferment are 350rpm, speed drop to 100～200rpm will ferment after 30 hours, make dissolved oxygen be controlled at 5%～30%, promptly adopt segmentation of the present invention to stir fermentation method.The glucose total amount will remain glucose and add in the fermentor tank under 20% prerequisite in the fermenting process after preceding 30 hours control glucose concn are 5%～10%, 30 hours simultaneously, add the KH of 1.0g/L simultaneously
2PO
4
B, fermentation were carried out 110 hours altogether, and glucose transforms fully, and the high performance liquid chromatography area normalization is analyzed in the fermented liquid and contained glycerine 2.5g/L as shown in Figure 5, erythritol 17g/L, and ribitol 86g/L, ribitol production process inversion rate of glucose is 43%.
Embodiment 4: as shown in Figure 2.Its processing step is:
(1) bacterial classification is selected for use.With embodiment 1.
(2) medium preparation.With embodiment 1.
(3) strain preparation.With embodiment 1.
(4) seed culture.With implementing 1.
(5) fermentation culture.With embodiment 3.
(6) fermented liquid is purified.
With step 5 gained fermented liquid (wherein the concentration of ribitol is 86g/L).Successively according to following steps purify (as accompanying drawing 3):
A, be that centrifugal 10min removes thalline under the 5000g condition at centrifugal force with fermented liquid.
B, centrifugal gained clear liquid add 5/1000ths gac, stir 30min and decolour under 50～80 ℃ of temperature condition, and suction filtration is removed gac then.
C, under decompression and 60 ℃ of temperature condition, it is more than 60% that the feed liquid after the decolouring is concentrated into ribitol content.
D, the concentrated solution after above-mentioned steps c concentrated place Ca type strong acid cation exchange resin column to advance to separate (PUROLITE PCR642 calcium type ion exchange resin); Its resin column length is 1000mm, and column internal diameter is 45mm, uses the deionized water wash-out after sample finishes on the fermented liquid.
E, the elutriant that contains ribitol that the d step is prepared receive merging, evaporate to dryness under reduced pressure, and treatment temp is controlled at below 65 ℃.
F, with step e resulting product, in menstruum ethanol, carry out recrystallization, after the vacuum-drying, products obtained therefrom purity is 99%, yield is more than 95%.Product detects add their confirmation (as accompanying drawing 6) through liquid chromatography and infrared spectra.Wherein wave number is a hydroxyl peak about 3400, and wave number is a methylene radical about 2900, and wave number 1050 and 1100 explanations have primary alconol and secondary alcohol groups to exist.The product infared spectrum meets the ribitol structure fully.
Lab scale effect of the present invention shows, has realized the original intention of its invention.
Claims (4)
1. preparation technology who adopts fermentation method to prepare ribitol, with glucose is raw material, oozing yeast bulb three type spore bacterium (Trichosporonoides oedocephalis) Atcc 16958 with height is starting strain, successively according to strain preparation, seed culture and three steps of fermentation are carried out, and it is characterized in that:
A, described fermentation step are seed liquor that the seed culture step is prepared with the fermention medium that contains glucose by 1～10% volume ratio, be inoculated in the sterilized fermentor tank that described fermention medium is housed, implement to stir fermentation culture;
B, described fermentation culture are to be 0～1Mpa at tank pressure, and temperature is 25～40 ℃, and dissolved oxygen is 5～95%, and PH carries out under 4～7 the processing condition; The time of fermentation culture process is 80～160h, to the residual sugar amount≤0.3% o'clock fermentation ends of its glucose;
The stirring of c, described fermenting process is that segmentation is stirred; The mixing speed of preceding 30h is controlled in 200～600rpm scope, and dissolved oxygen is controlled in 65～95% scopes; Mixing speed behind the 30h is controlled in 100～300rpm scope, and dissolved oxygen is controlled in 5～65% scopes.
2. employing fermentation method according to claim 1 prepares the preparation technology of ribitol, it is characterized in that, the glucose in the described fermention medium, and segmentation adds in the described fermentor tank in the fermentation culture process; In earlier fermentation 0～30h time range, the control glucose concn is in 5～15% scopes; Behind 30h, add glucose and KH
2PO
4, and control glucose concn in 5～25% scopes, described KH
2PO
4Add-on be controlled in 0.1～1.5g/L scope.
3. employing fermentation method according to claim 1 and 2 prepares the preparation technology of ribitol, it is characterized in that, behind fermentation step, also comprises purification step; Described purification step is the fermented liquid that will produce by fermentation step, removes thalline through centrifugation, activated carbon decolorizing, underpressure distillation concentrates, chromatographic separation resin chromatography, chromatographic solution concentrates and the organic solvent recrystallization, and gets the ribitol product of high purity white crystals.
4. employing fermentation method according to claim 3 prepares the preparation technology of ribitol, it is characterized in that, described chromatographic separation resin is a Ca type storng-acid cation exchange resin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100235961A CN101260414B (en) | 2008-04-11 | 2008-04-11 | Technique for preparing ribitol by fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100235961A CN101260414B (en) | 2008-04-11 | 2008-04-11 | Technique for preparing ribitol by fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101260414A CN101260414A (en) | 2008-09-10 |
| CN101260414B true CN101260414B (en) | 2010-12-08 |
Family
ID=39961112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100235961A Active CN101260414B (en) | 2008-04-11 | 2008-04-11 | Technique for preparing ribitol by fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101260414B (en) |
-
2008
- 2008-04-11 CN CN2008100235961A patent/CN101260414B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| JP特开平10-210994A 1998.08.11 |
| JP特开平11-332592A 1999.12.07 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101260414A (en) | 2008-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101736042B (en) | Method for producing L-lactic acid | |
| CN102268490B (en) | Clean technique for co-producing xylose, xylitol and arabinose from agricultural waste and forest waste | |
| CN101748161B (en) | Succinic acid purification process through anaerobic fermentation | |
| US20110183394A1 (en) | Method of producing yeast biomass | |
| CN101538589A (en) | New clean method for producing xylitol and arabinose | |
| WO2017035525A1 (en) | Methods for the digestion of soluble components isolated from the spent grains of a fermentation process | |
| CN101254969B (en) | Method for preparing microbial flocculant by using byproduct bacterial of fermentation industry | |
| CN101705253B (en) | Method for treating xylose mother solution | |
| US11866756B2 (en) | Methods for co-producing erythritol and arabinose by using xylose mother liquor | |
| CN115354064B (en) | Method for producing medium-chain fatty acid by two-phase partition of anaerobic dry fermentation | |
| CN101260414B (en) | Technique for preparing ribitol by fermentation method | |
| FI85501B (en) | FOERFARANDE FOER FRAMSTAELLNING AV POLYOLER GENOM PAO INDUSTRIELL SKALA BASERAD FERMENTATION AV SOCKER. | |
| CN101054597A (en) | Method for producing cystine by microorganism transformation | |
| CN110372606B (en) | Method for separating and purifying cytosine from microbial fermentation liquor | |
| RU2405829C2 (en) | Method of preparing organic solvents | |
| CN108486173B (en) | Preparation method of alpha-ketoglutaric acid | |
| CN101294176B (en) | Method for preparing L-ribose with ribitol fermentation liquor biotransformation | |
| CN102634463B (en) | Saccharomycete producing xylitol and applicaton of saccharomycete | |
| CN115044489B (en) | Method for culturing saccharomyces cerevisiae by utilizing continuous fermentation and separation integrated technology | |
| CN101343643B (en) | High production method for glycerol with multi-step zymotechnics | |
| RU2329302C2 (en) | Method of fermenting molasses wort | |
| CN112746088A (en) | Method for co-producing xylitol and fuel ethanol by fermenting lignocellulose serving as raw material | |
| RU2643255C2 (en) | Method of feed yeast production from peat | |
| CN116083500B (en) | Process for continuous production of erythrose | |
| GB2065699A (en) | Ethanol production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |