CN102796797B - Method for preparing xylitol and its intermediate D-xylosone by microbial transformation of glucose and strain used in the same - Google Patents
Method for preparing xylitol and its intermediate D-xylosone by microbial transformation of glucose and strain used in the same Download PDFInfo
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- CN102796797B CN102796797B CN201210281967.2A CN201210281967A CN102796797B CN 102796797 B CN102796797 B CN 102796797B CN 201210281967 A CN201210281967 A CN 201210281967A CN 102796797 B CN102796797 B CN 102796797B
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Abstract
The invention discloses a method for preparing D-xylosone by microbial transformation of glucose, comprising the following steps: firstly, respectively preparing an osmophilic yeast seed liquid and a gluconobacter oxydans seed liquid by using glucose as a raw material, then inoculating the osmophilic yeast seed liquid to conduct arabitol fermentation, and then in middle and later stage inoculating the gluconobacter oxydans seed liquid to conduct mixed culture fermentation, simultaneously controlling the glucose content in the broth to be 5-10g/L, and converting arabitol into D-xylosone. According to the method, the feedback inhibition of arabitol is removed, and the efficiency of preparing D-xylosone from glucose is raised. The invention further discloses a method for preparing xylitol by microbial transformation of glucose, comprising the following steps: firstly converting glucose into D-xylosone by the above method, then converting D-xylosone into D-xylose by isomerization of enzyme, extracting and refining, and conducting catalytic hydrogenation to obtain xylitol. According to the method, the production efficiency of xylitol is raised, and the cost of preparing xylitol by biological method is reduced.
Description
Technical field
The present invention relates to a kind of Xylitol intermediate---the preparation method of D-xylulose, utilize this intermediate to prepare method and the bacterial classification used of Xylitol, be specifically related to a kind ofly prepare the method for D-xylulose and Xylitol and bacterial classification used take glucose as raw material by microbial transformation.
Background technology
Xylitol belongs to five carbon polyols, is a kind of natural sweeteners.Xylitol is not utilized by bacterium in oral cavity, has good preventing caries function.On the other hand, Xylitol participates in without Regular Insulin, can directly enter human body cell and carry out metabolism supplementing energy, can not cause blood sugar increasing, can alleviate the many foods of the many drinks of diabetics diuresis symptom.Xylitol sugariness and sucrose are suitable, but have the calorie lower than sucrose; Compared with other sugar alcohols, it has the highest solution heat, and edible have refrigerant sense, and therefore Xylitol is a kind of desirable sucrose substitute, is widely used in recent years chewing gum, toothpaste, sugarfree foods, medicine and other fields.
The production method of Xylitol can be divided into following two kinds at present:
1. chemical synthesis: the method that is industrial conventional production Xylitol, its ultimate principle is that pentosan obtains wood sugar through acid hydrolysis, wood sugar hydrogenation under the katalysis of nickel generates Xylitol, although the method raw material is easy to get, but its complex process, by product is many, separating-purifying is difficult, consume a large amount of soda acids in, process high to equipment requirements and produce a large amount of wastes, contaminate environment is serious, cause current Xylitol price higher, this has also limited the widespread use of Xylitol to a certain extent.
2. biotransformation method: be divided into (1) take wood sugar as raw material according to raw material difference: by agricultural wastes if the xylan in corn cob, straw, bagasse etc. is after dilute acid hydrolysis, obtain the hydrolyzed solution that primary product is wood sugar, then utilize the wood sugar in microorganism fermentation hydrolysis liquid to generate Xylitol, but this method fermented liquid Determination of Xylitol is very low, separation and Extraction cost is still very high, does not have industrialization meaning; (2) take glucose as raw material: D-Glucose changes into D-R alcohol under the effect of osmophilic yeast, then D-R alcohol is oxidized to D-xylulose under the effect of gluconobacter oxydans, last D-xylulose generates Xylitol by the reductive action of yeast (Candida guilliermodii), is called again three step fermentation methods.But in this method the first step fermentation, product arabitol has obvious product and suppresses phenomenon, causes pectinose determining alcohol on the low side, and the 3rd step yeast conversion D-xylulose is that Xylitol efficiency is lower in addition, and production concentration is no more than 30g/L.Therefore the problem that, Bioconversion of Xylitol mainly exists is at present: conversion of glucose generates arabitol and D-xylulose transforms production Xylitol inefficiency.
Patent EP403392A and patent EP421882A disclose such method, first produce arabitol with osmophilic yeast, then by the bacterium that adopts acetobacter, Gluconobacter, klebsiella, arabitol is converted into D-xylulose, transforming D-xylulose by the effect of glucose (wood sugar) isomerase is the mixed solution of D-wood sugar and D-xylulose, above mixed solution hydrogenation obtain Xylitol.Although it is the problem of Xylitol inefficiency that this method has solved biological process conversion D-xylulose, osmophilic yeast generates arabitol low efficiency problem and still exists, and makes the method production cost still higher than chemical synthesis.
In bio-transformation glucose production Xylitol technique, adopt osmophilic yeast transforming glucose to produce arabitol, arabitol can produce and suppress phenomenon thalline, causes middle and later periods arabitol formation speed to reduce.Osmophilic yeast transforming glucose is arabitol optimal pH 4.5-5.5, and temperature is 30-35 ℃, needs yeast extract paste, MgSO
4, KH
2pO
4deng as nutritive ingredient; And gluconobacter oxydans conversion arabitol generation D-xylulose speed is very fast, the about 20-30 h of 150 g/L arabitol can transform, optimal pH 5.0-5.5, and temperature is 30-35 ℃, needs yeast extract paste, MgSO
4, KH
2pO
4deng as nutritive ingredient.As can be seen from the above the growth of osmophilic yeast and gluconobacter oxydans and biotransformation condition are basically identical, utilize this characteristic, in patent CN200410031949.4, attempt adopting microorganism mixed fungus fermentation to generate the method for Xylitol, the method is first cultivated respectively yeast saccharomyces cerevisiae and gluconobacter oxydans kind liquid, then by both while inoculation fermentation liquid mixed culture, substrate glucose is about 160 g/L, and final fermented liquid Xylitol concentration is about 30 g/L, and transformation efficiency is still very low.
In sum, chemical synthesis is produced Xylitol, and soda acid and energy consumption consumption are larger, and environmental pollution is serious; Biotransformation method is produced Xylitol, and mild condition, has fewer environmental impacts, and is the trend of following Xylitol production technology, but because its efficiency of producing Xylitol is low, does not have industrial value.
Summary of the invention
The present invention is directed to the defect that conversion of glucose in prior art becomes arabitol inefficiency, provide a kind of microbial transformation glucose to prepare the method for D-xylulose, the method can be removed the feedback inhibition of arabitol, and the productive rate of final D-xylulose is effectively improved.
The present invention also provides the method for utilizing D-xylulose that aforesaid method makes to prepare Xylitol, and the method gained Xylitol productive rate significantly improves.
The present invention also provides the osmophilic yeast bacterial classification that transforming glucose is arabitol---Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 CGMCC NO.6197.
Contriver studies by experiment and finds that the efficiency of osmophilic yeast transforming glucose in early stage generation arabitol is higher, along with the increase of pectinose determining alcohol, conversion rate declines, if add the arabitol of high density early stage, it is also very slow that osmophilic yeast transforming glucose in early stage generates arabitol speed, shows that arabitol has feedback inhibition to osmophilic yeast; On the other hand, gluconobacter oxydans is under high glucose concentration conditions, and the speed that transforms arabitol generation D-xylulose is very slow, analyzes and finds that above factor is to cause osmophilic yeast and the not high reason of gluconobacter oxydans mixed fungus fermentation efficiency.In research process, contriver has attempted first cultivating osmophilic yeast glucose fermentation and has been converted into arabitol, middle and later periods inoculation gluconobacter oxydans and the mode of control glucose concn below 10 g/L are prepared D-xylulose, the feedback inhibition of arabitol is adopted mixed fungus fermentation in this way and can be removed in discovery, significantly improves the efficiency that conversion of glucose is D-xylulose.Contriver is further studied, and has obtained producing the technical scheme of D-xylulose, as follows:
A kind of microbial transformation glucose is prepared the method for D-xylulose, take D-Glucose as raw material, by can transforming glucose the osmophilic yeast that is arabitol and can transform the gluconobacter oxydans that arabitol is D-xylulose (Gluconobacter oxydans) mixed fungus fermentation and must contain D-xylulose fermented liquid, fermented liquid is through further processing to obtain D-xylulose, it is characterized in that, mixed fungus fermentation comprises the following steps: first, prepare respectively osmophilic yeast kind liquid and gluconobacter oxydans kind liquid, then first osmophilic yeast kind liquid is inoculated into containing carrying out fermentation culture in the fermention medium of glucose, be arabitol by conversion of glucose, again gluconobacter oxydans kind liquid is inoculated in fermented liquid in the osmophilic yeast fermentation middle and later periods, in controlled fermentation liquid, glucose content is 5-10 g/L simultaneously, continue fermentation arabitol is converted into D-xylulose, must contain the fermented liquid of D-xylulose.
The main innovate point of above-mentioned preparation D-xylulose method is by original two kinds of bacteria culture fluids being added containing carrying out mixed fungus fermentation in dextrose culture-medium simultaneously by the method for mixed fungus fermentation, the mode that makes conversion of glucose become arabitol and arabitol to be converted into D-xylulose becomes that first in glucose, to inoculate osmophilic yeast transforming glucose be arabitol, inoculating gluconobacter oxydans in the fermentation middle and later periods ferments arabitol is converted into D-xylulose, the concentration 10g/L of glucose left and right in control process, remove the feedback inhibition of arabitol, to improve the transformation efficiency of arabitol, improve the productive rate of D-xylulose.
Fig. 2 is that microorganism mixed fungus fermentation is produced Xylitol intermediate (D-xylulose) canonical process graphic representation, as can be seen from the figure: after osmophilic yeast inoculation, OD value (biomass) increases sharply, and when 20 h, enters stationary phase, meanwhile, arabitol starts to generate; At 20-80 h, thalli growth is slow, and glucose consumes fast, and arabitol generates fast; After 80 h inoculation gluconobacter oxydans, there is diauxic growth in thalline, arabitol content declines rapidly, when 100 h, drop to 3 g/L, keep ever since lower level, in process, D-xylulose content raises rapidly, until OD value declines after fermentation 140 h, D-xylulose throughput rate declines gradually, fermentation 150 h, D-xylulose content 148 g/L in fermented liquid.
In aforesaid method, osmophilic yeast used and gluconobacter oxydans can be disclosedly in prior art to can be used for all osmophilic yeast bacterial classifications and the gluconobacter oxydans bacterial classification that conversion of glucose is D-xylulose, for example, in patent EP403392A, patent EP421882A and patent CN200410031949.4 disclosed bacterial classification, as long as carry out according to mode of the present invention, mixed fungus fermentation all can play releasing arabitol feedback inhibition, raising conversion of glucose is the object of D-xylulose efficiency.Using the disclosed bacterial classification of prior art, its incubation time and culture condition can be realized according to the disclosed content of prior art.
Further, in aforesaid method, can transforming glucose be arabitol osmophilic yeast preferentially selects contriver from the Ao Mo of row filter Kodak yeast (Kodamaea ohmeri) TL-1123, this TL-1123 bacterial classification is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation date is on June 6th, 2012, and deposit number is CGMCC NO.6197.This bacterial classification can resistance to height oozes, high temperature resistant, can normal growth breeding under 200 g/L glucose and 35 ℃ of conditions, can generate arabitol by Efficient Conversion glucose, transformation efficiency can reach 44%, in fermented liquid, the content of D-xylulose can reach 150 g/L.This bacterial screening process is:
1. by samples such as peach, pumpkin, Pulp Citrulli Pollens, under aseptic condition, access (2 × 25 cm test tubes, liquid amount 10 mL) in enrichment medium and, in 35 ° of C, under 220 r/min, cultivate 2 ~ 4 d.Enrichment medium composition (gL
-1): glucose 50, yeast extract paste 3, peptone 3.
2. move transfer enrichment medium that 10 mL are housed of the nutrient solution in is 1. shaken in pipe and cultivated 1 ~ 3 day with 10% inoculum size with liquid-transfering gun, so repeat 3 ~ 4 times, bacterium liquid dilution spread is carried out to plate separation in isolation medium, under 35 ° of C, cultivate 2 ~ 4 d.Plate isolation and slant preservation substratum composition (gL
-1): enrichment medium+agar 20.
3. the single bacterium colony enlarged culturing obtaining will be separated, under 30 ° of C, cultivate after 2 ~ 4 d, the triangular flask interior (250 mL shaking flasks fill 50 mL fermented liquids) of 50 mL primary dcreening operation fermentation cultures is equipped with in access respectively, under 35 ° of C, cultivate 3 d, after fermentation culture finishes, detect D-R alcohol content, leave and take the bacterial strain that productive rate is higher and carry out multiple sieve.Primary dcreening operation fermention medium composition (gL
-1): glucose 50, yeast extract paste 3, peptone 3, CaCO
315.
4. the access of multiple sieve bacterial strain is equipped with in the 250 mL triangular flasks that 50 mL sieve fermentation culture again, under 35 ° of C, cultivate 3 d, after fermentation culture finishes, detect D-R alcohol content, choose the bacterial strain that arabitol productive rate is high and carry out further fermenting experiment and strain identification experiment.Sieve again fermention medium composition (gL
-1): glucose 200, yeast extract paste 3, peptone 3, CaCO
315.
Further, in aforesaid method, can transform arabitol and be disclosed gluconobacter oxydans (Gluconobacter oxydans) NH-10 in the preferred patent CN200910024579.4 of gluconobacter oxydans of D-xylulose, preserving number: CGMCC No.2709.
Further, in aforesaid method, mixed fungus fermentation specifically comprises the following steps: first, prepare respectively osmophilic yeast kind liquid and gluconobacter oxydans kind liquid, the inoculum size of then pressing 5-15%, is first inoculated into osmophilic yeast kind liquid in the fermention medium that contains glucose sugar, at 100-200 rpm, ventilation ratio is 0.3-0.8 L/Lmin, and 30-35 ℃ of condition bottom fermentation cultivated 65-85 h, is arabitol by conversion of glucose, and then gluconobacter oxydans kind liquid is inoculated in fermented liquid by the inoculum size of 5-15%, at 100-200 rpm, ventilation ratio is 0.3-0.8 L/Lmin, under 30-35 ℃ of condition, continue fermentation, simultaneously in fermented liquid, stream adds glucose and alkaline substance solution (alkaline substance solution is sodium hydroxide, potassium hydroxide, sodium carbonate, the aqueous solution of salt of wormwood etc.), making glucose content in fermented liquid is 5-10 g/L, pH is 4.6-5.4, whole fermentation time continues to stop supplementing glucose after 130-150 h, after exhausting, glucose and arabitol stop fermentation, must contain the fermented liquid of D-xylulose.
Further, fermention medium consists of: glucose 120-160 g/L, corn steep liquor 5-10 g/L, yeast extract paste 3-5 g/L, KH
2pO
43-4 g/L, MgSO
40.5-1.5 g/L, pH 5.0-5.5.
Further, osmophilic yeast seed liquor adopts following method preparation: get the osmophilic yeast bacterial classification that a transfering loop inclined-plane is preserved, be seeded in seed culture medium A, under 100-250 rpm, 30-35 ℃ condition, cultivate 10-20 h, obtain one-level kind liquid, gained one-level kind liquid is inoculated in seed culture medium A by the inoculum size of 5-15%, is enlarged culturing 12-20 h under 0.3-0.8 L/Lmin, 30-35 ℃ condition at 100-200 rpm, ventilation ratio, obtains osmophilic yeast kind liquid; Seed culture medium A consists of: glucose 60-80 g/L, corn steep liquor 5-10 g/L, yeast extract paste 3-5 g/L, MgSO
40.5-1.5 g/L, natural pH;
Gluconobacter oxydans seed liquor adopts following method preparation: the whole thalline on a gluconobacter oxydans inclined-plane are inoculated in seed culture medium B, under 100-250 rpm, 30-35 ℃ condition, cultivate 16-24 h, obtain one-level kind liquid, gained one-level kind liquid is inoculated in seed culture medium B by the inoculum size of 5-15%, be enlarged culturing 16-20 h under 0.3-0.8 L/Lmin, 30-35 ℃ condition at 100-200 rpm, ventilation ratio, obtain gluconobacter oxydans kind liquid; Seed culture medium B consists of: arabitol 100-120 g/L, yeast extract paste 5-10 g/L, calcium carbonate 0.5-1.5 g/L, pH 5.1-5.4.
Further, in aforesaid method, last handling process containing D-xylulose fermented liquid is: fermented liquid is removed to thalline and impurity through Plate Filtration, ultra-filtration membrane ultrafiltration, activated carbon decolorizing and ion-exchange, then concentrated, crystallization obtains D-xylulose, be more specifically: fermented liquid is adopted to Plate Filtration degerming, is the ultrafiltration membrance filter of 1000-3000 Da with molecular weight cut-off again, then use decolorizing with activated carbon and ion exchange treatment.Fermented liquid after ion-exchange being concentrated into dry matter content is again 80-95%, then be cooled to 40-55 ℃, in concentrated solution, add the D-xylulose of dry matter content 2-4% as crystal seed, be more naturally cooled to room temperature and carry out crystallization, centrifugal after crystallization, obtain D-xylulose.
The above-mentioned D-of making xylulose is intermediate prepared by Xylitol, the D-xylulose that can make with aforesaid method is further prepared Xylitol, step is: take glucose sugar as raw material, the D-xylulose making by the method for above-mentioned mixed fungus fermentation further adopts prior art to be reduced to Xylitol, to obtain final product.
The technology that D-xylulose is reduced to Xylitol has a lot, for example can adopt the reductive action of yeast (Candida guilliermodii) that D-xylulose is reduced to Xylitol, also can adopt xylose isomerase (to be also glucose isomerase, lower same) first transform the mixed solution that D-xylulose is D-wood sugar and D-xylulose, then the separation of D-wood sugar, hydrogenation are obtained to Xylitol.Above-mentioned prior art all can be used for, in the present invention, D-xylulose is reduced to Xylitol, but preferably adopts xylose isomerase enzymatic conversion D-xylulose to generate D-wood sugar, adopts xylose isomerase gained Xylitol efficiency higher, is more beneficial to industrialized production.
The method that the present invention prepares Xylitol preferably adopts mixed fungus fermentation to prepare D-xylulose and adopts the mode of the two combination of xylose isomerase enzymatic conversion D-xylulose to carry out, and gained Xylitol productive rate improves greatly, and preferred method scheme is as follows:
A kind of microbial transformation glucose is prepared the method for Xylitol, take glucose sugar as raw material, the method that adopts mentioned microorganism transforming glucose to prepare D-xylulose is D-xylulose by conversion of glucose, adopt xylose isomerase D-xylulose to be converted into the mixed solution of D-wood sugar and D-xylulose, then the separation of D-wood sugar, hydrogenation are obtained to Xylitol, comprise the following steps:
1. the preparation of D-xylulose: the method that adopts the mixed bacterium cultivation of mentioned microorganism transforming glucose to prepare D-xylulose is prepared D-xylulose;
2. the preparation of D-wood sugar: the D-xylulose by step in is 1. made into the aqueous solution, makes the isomery liquid glucose that contains D-xylulose and D-wood sugar under the effect of xylose isomerase;
3. the extraction of D-wood sugar is refining: utilize the isomery liquid glucose of simulation moving-bed device treatment step in 2., D-xylulose is separated with D-wood sugar and obtain being rich in D-xylulose component and be rich in D-wood sugar component, to be rich in D-wood sugar component crosses ion exchange resin and removes impurity, then concentrated, crystallization, obtains D-wood sugar; Being rich in D-xylulose component enters step and applies mechanically in 2.;
4. the preparation of Xylitol: the D-wood sugar by step in is 3. made into the aqueous solution is carried out hydrogenation reaction under catalyzer exists, and obtains xylitol solution;
5. the extraction of Xylitol is refining: Xylitol reacting liquid filtering is removed to catalyzer, crossed ion exchange resin and remove impurity, and then concentrated, crystallization, obtains D-Xylitol.
Further, the preparation of D-wood sugar specifically comprises the following steps: the D-xylulose by step in is 1. made into the aqueous solution of 35-45%, and adjusting pH is 8.0-8.3, then adds magnesium sulfate and SO in the aqueous solution
2, the concentration to magnesium sulfate in the aqueous solution is 43-48 mg/L, SO
2concentration in the aqueous solution is 95-120 mg/L, then by the D-xylulose aqueous solution with 2-4 m
3/ h flow velocity flows through enzyme isomery post, and isomery post is containing xylose isomerase 200-400 kilogram, and temperature of reaction 55-60 ℃ must contain the isomery liquid glucose of D-xylulose and D-wood sugar;
Further, the extraction of D-wood sugar is refined and is specifically comprised the following steps: using calcium type Zeo-karb as sorbent material, take water as eluent, at 50-65 ℃, utilize the isomery liquid glucose of simulation moving-bed device treatment step in 2., obtain being rich in D-xylulose component and be rich in D-wood sugar component, to be rich in wood sugar component and carry out in the usual way activated carbon decolorizing, Plate Filtration and excessively ion exchange resin are removed impurity, it is more than 75-90% being concentrated into dry matter content, then be naturally cooled to 40-60 ℃, in concentrated solution, add the D-wood sugar of dry matter content 3-5% as crystal seed, naturally be cooled to again room temperature and carry out crystallization, after crystallization, crystal is centrifugal, obtain D-wood sugar, being rich in D-xylulose component enters step and applies mechanically in 2..The ion exchange resin using while removing impurity has cationic ion exchange resin and anionic ion exchange resin, wherein, cationic ion exchange resin preferred large porous strong acid vinylbenzene-divinyl benzene series resin and macropore weak acid vinylformic acid-divinyl benzene series resin, anionic ion exchange resin preferred macropore weak base phenylethylene resin series and macropore strong base phenylethylene resin series.
Further, the preparation of Xylitol specifically comprises the following steps: the D-wood sugar by step in is 3. made into the aqueous solution of 40-50%, add the Raney's nickel of aqueous solution weight 0.3-0.8% or thunder Buddhist nun ruthenium as catalyzer, at 140-170 ℃, 7.2-8.0 carry out hydrogenation reaction under Mpa hydrogen pressure, reaction 2-4 h, obtains xylitol solution.
Further, the extraction of Xylitol is refined and is specifically comprised the following steps: xylitol solution is carried out in the usual way to activated carbon decolorizing, Plate Filtration and crosses ion exchange resin and remove impurity, it is more than 75-90% being concentrated into dry matter content, then be naturally cooled to 40-60 ℃, in concentrated solution, add the Xylitol of dry matter content 2-4% as crystal seed, be then cooled to room temperature and carry out crystallization, after crystallization, crystal is centrifugal, 55-75 ℃ of oven dry, obtains D-Xylitol.The ion exchange resin using while removing impurity has cationic ion exchange resin and anionic ion exchange resin, wherein, cationic ion exchange resin preferred large porous strong acid vinylbenzene-divinyl benzene series resin and macropore weak acid vinylformic acid-divinyl benzene series resin, anionic ion exchange resin preferred macropore weak base phenylethylene resin series and macropore strong base phenylethylene resin series.
The present invention has the following advantages:
1, the mixed fungus fermentation technology of preparation D-xylulose is improved, first inoculate osmophilic yeast and carry out arabitol fermentation, then inoculate gluconobacter oxydans in the fermentation middle and later periods, remove the feedback inhibition of arabitol, higher more simultaneously than the method transformation efficiency of inoculating osmophilic yeast and gluconobacter oxydans in prior art, improve the productive rate of D-xylulose; One-step fermentation can be D-xylulose by conversion of glucose, and than first preparing arabitol with osmophilic yeast in prior art, then it is simple to operate to prepare the two-step fermenting of D-xylulose with arabitol, and the cycle is short, has saved raw material, has reduced energy consumption.
2, preferably use from the Ao Mo of row filter Kodak yeast (Kodamaea ohmeri) TL-1123 as osmophilic yeast, the resistance to height of this bacterial strain oozes, high temperature resistant, normal growth breeding under 200 g/L glucose and 35 ℃ of conditions, can generate arabitol by Efficient Conversion glucose, transformation efficiency can reach 44%, and in fermented liquid, the content of D-xylulose can reach 150 g/L.
3, utilize D-xylulose that mixed fungus fermentation method makes to prepare Xylitol and improved production efficiency, reduced cost.Further, preferably adopt xylose isomerase to prepare the method for Xylitol, efficiency further improves, and is easier to operation and realizes in actual production.
Accompanying drawing explanation
For content of the present invention is more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein:
Fig. 1 is that microbial transformation glucose of the present invention is prepared Xylitol and intermediate D-xylulose process flow sheet thereof;
Fig. 2 is that microorganism mixed fungus fermentation is produced Xylitol intermediate (D-xylulose) canonical process graphic representation.
preservation information
The present invention osmophilic yeast Ao Mo used Kodak yeast (Kodamaea ohmeri) TL-1123 is in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, and preservation date is on June 6th, 2012, and deposit number is CGMCC NO.6197.
Embodiment
According to following embodiment, the present invention may be better understood.But, those skilled in the art will readily understand, the described concrete technology condition of embodiment, material proportion and result thereof be only for the present invention is described, and should also can not limit the present invention described in claims.
In following embodiment, osmophilic yeast used is Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123, deposit number: CGMCC NO.6197; Gluconobacter oxydans used is gluconobacter oxydans (Gluconobacter oxydans) NH-10, deposit number: CGMCC NO.2079, and open in patent CN200910024579.4.Therefore, the present invention's bacterial classification used meets and discloses sufficient requirement.Ao Mo Kodak yeast and gluconobacter oxydans are preserved at 4 ℃ with inclined-plane, Ao Mo Kodak yeast preservation slant medium composition (g/L): glucose 50, yeast extract paste 3, peptone 3, agar 20, natural pH; Gluconobacter oxydans preservation slant medium composition (g/L): arabitol 80, yeast extract paste 5, peptone 3, calcium carbonate 1, agar 20, pH 5.2.
In following embodiment, removing impurity Zeo-karb used is the mixture of Amberlite IRC50 and Amberlite FPC22Na, anionite-exchange resin is the mixture of Amberlite FPA51 and Amberlite FPA90CI, xylose isomerase, also referred to as glucose isomerase, adopt the conventional fixed glucose isomerase of sugar alcohol industry.
embodiment 1
(1) substratum: seed culture medium A consists of: glucose 80 g/L, corn steep liquor 10 g/L, yeast extract paste 3 g/L, MgSO
40.5 g/L, natural pH; Seed culture medium B consists of: arabitol 120 g/L, yeast extract paste 10 g/L, calcium carbonate 1.5 g/L, pH 5.2; Fermention medium consists of: glucose 160 g/L, corn steep liquor 10 g/L, yeast extract paste 3 g/L, KH
2pO
43 g/L, MgSO
40.5 g/L, pH 5.0.
(2) seed liquor preparation: a) Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 seed liquor: get a ring bacterium from inclined-plane, be seeded in (culture medium A) in shaking flask, liquid amount 15%, 250 rpm, cultivate 16 h for 35 ℃ and (be primary seed solution as yeast shaking flask kind liquid, lower same), then by 10% inoculum size, yeast shaking flask kind liquid is seeded in 100 L fermentor tank kind liquid (culture medium A), liquid amount 60%, ventilation ratio is 0.5 L/Lmin, 150 rpm, 35 ℃ of enlarged culturing 14 h (are osmophilic yeast seed liquor as yeast kind of the liquid that spreads cultivation, lower same), b) gluconobacter oxydans seed liquor: the whole bacterium colonies of wash-out from a gluconobacter oxydans inclined-plane, be seeded in (substratum B) in shaking flask, liquid amount 15%, 250 rpm, cultivate 16 h for 35 ℃ and (be primary seed solution as gluconobacter oxydans shaking flask kind liquid, lower same), then by 10% inoculum size, gluconobacter oxydans shaking flask kind liquid is seeded in 100 L fermentor tank kind liquid (substratum B), liquid amount 60%, ventilation ratio is 0.5 L/Lmin, 200 rpm, 35 ℃ of enlarged culturing 16 h (are gluconobacter oxydans seed liquor as gluconobacter oxydans kind of the liquid that spreads cultivation, lower same).
(3) mixed fungus fermentation: by 10% inoculum size, kind of the liquid that first yeast spread cultivation is seeded in 1m
3in fermentor tank (fermention medium), liquid amount 60%, 200 rpm, ventilation ratio is 0.5 L/Lmin, and 35 ℃ of fermentation culture 85 h, then by 10% inoculum size, kind of the liquid that again gluconobacter oxydans spread cultivation is seeded in fermented liquid and continues mixed fungus fermentation, and in process, stream adds glucose and 10wt%Na
2cO
3solution, 150 rpm, ventilation ratio is 0.5 L/Lmin, and in controlled fermentation liquid, glucose content is at 5 g/L, and pH is 5.1,145 h(yeast fermentations+gluconobacter oxydans fermentation 145h altogether ferments, lower same), stop mending sugar, treat that glucose and arabitol exhaust lower tank, in Liquid Detection fermented liquid, the content of D-xylulose is 148.3 g/L, and glucose is 43.2% to the transformation efficiency of D-xylulose.
(4) extraction of D-xylulose is refining: first fermented liquid is adopted to the degerming of usual manner Plate Filtration, then to adopt molecular weight cut-off be the ultrafiltration membrance filter of 1000 Da, then carry out decolorizing with activated carbon and ion-exchange.Reconcentration to dry matter content is 80%, is then cooled to 40 ℃, in concentrated solution, adds the D-xylulose of dry matter content 2% as crystal seed, is more naturally cooled to room temperature and carries out crystallization, centrifugal after crystallization, obtains D-xylulose.
(5) enzymatic conversion: refining D-xylulose is made into the aqueous solution of 35-45%, adjusting pH is 8.0-8.3, then adds magnesium sulfate and SO in the aqueous solution
2, the concentration to magnesium sulfate in the aqueous solution is 48 mg/L, SO
2concentration in the aqueous solution is 95 mg/L, and the D-xylulose aqueous solution is with 2 m
3/ h flow velocity flows through enzyme isomery post, and isomery post is containing 300 kilograms of xylose isomerases, and 60 ℃ of temperature of reaction must contain the isomery liquid glucose of D-xylulose and D-wood sugar; (Liquid Detection contains D-xylulose 43.5% and D-wood sugar 55.2%, oligosaccharides 1.3%).
(6) wood sugar extracts refining: utilize the simulation moving-bed device of 12 post to process isomery liquid glucose, separate D-xylulose and D-wood sugar; Using calcium type Zeo-karb as sorbent material, take water as eluent, continuously feeding at 65 ℃, water inlet, discharging, obtain being rich in D-xylulose component (D-xylulose purity 91.1%, concentration 25.56%) and be rich in D-wood sugar (D-wood sugar purity 95.3%, concentration 29.65%) component; To be rich in wood sugar component and cross in a usual manner ion exchange resin, and remove impurity, being then concentrated into dry matter content is 80%, naturally be cooled to 50 ℃, to the wood sugar crystal seed that adds relative dry matter content 3% in concentrated solution, be naturally cooled to room temperature, centrifugal, obtain crystallization D-wood sugar; Being rich in xylulose component returns step (5) and carries out enzyme isomery.
(7) hydrogenation: crystalline xylose is made into 45% solution, and 0.3 % adds Raney's nickel with solution weight, at 150 ℃, under 8.0 Mpa hydrogen pressures, carries out hydrogenation reaction, and reaction times 3 h, obtains xylitol solution.
(8) extraction of Xylitol is refining: xylitol solution is carried out in the usual way to activated carbon decolorizing, Plate Filtration and crosses ion exchange resin and remove impurity, being concentrated into dry matter content is 84%, then be naturally cooled to 40 ℃, in concentrated solution, add the Xylitol of relative dry matter content 4% as crystal seed, then be naturally cooled to room temperature and carry out crystallization, after crystallization, crystal is centrifugal, 65 ℃ of oven dry, obtain D-Xylitol.
embodiment 2
(1) substratum: seed culture medium A consists of: glucose 70 g/L, corn steep liquor 8 g/L, yeast extract paste 4.5 g/L, MgSO
40.9 g/L, natural pH; Seed culture medium B consists of: arabitol 110 g/L, yeast extract paste 7 g/L, calcium carbonate 1.2 g/L, pH 5.1; Fermention medium consists of: glucose 150 g/L, corn steep liquor 8 g/L, yeast extract paste 4 g/L, KH
2pO
43.5 g/L, MgSO
40.9 g/L, pH 5.3.
(2) seed liquor preparation: a) Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 seed liquor: get a ring bacterium from inclined-plane, be seeded in (culture medium A) in shaking flask, liquid amount 15%, 180 rpm, cultivate 18 h as yeast shaking flask kind liquid, then by 15% inoculum size, yeast shaking flask kind liquid are seeded in 100 L fermentor tank kind liquid (culture medium A) for 33 ℃, liquid amount 60%, ventilation ratio is 0.8 L/Lmin, 100 rpm, and 33 ℃ of enlarged culturing 12 h are as yeast kind of the liquid that spreads cultivation; B) gluconobacter oxydans kind liquid: the whole bacterium colonies of wash-out from a gluconobacter oxydans inclined-plane, be seeded in (substratum B) in shaking flask, liquid amount 15%, 100 rpm, cultivate 20 h as gluconobacter oxydans shaking flask kind liquid, then by 15% inoculum size, gluconobacter oxydans shaking flask kind liquid are seeded in 100 L fermentor tank kind liquid (substratum B) for 33 ℃, liquid amount 60%, ventilation ratio is 0.8 L/Lmin, 100 rpm, and 33 ℃ of enlarged culturing 16 h are as gluconobacter oxydans kind of the liquid that spreads cultivation.
(3) mixed fungus fermentation: by 10% inoculum size, kind of the liquid that first yeast spread cultivation is seeded in 1m
3in fermentor tank (fermention medium), liquid amount 60%, 200 rpm, ventilation ratio is 0.3 L/Lmin, 33 ℃ of fermentation culture 75 h, then by 5% inoculum size, kind of the liquid that again gluconobacter oxydans spread cultivation is seeded in fermented liquid relaying supervention ferment, ventilation ratio is 0.8 L/Lmin, 100 rpm, in process, stream adds glucose and 10wt%NaOH solution, in controlled fermentation liquid, glucose content is at 8 g/L, pH is 4.6, 150 h ferment, stop mending sugar, treat that glucose and arabitol exhaust lower tank, in Liquid Detection fermented liquid, the content of D-xylulose is 153.2 g/L, glucose is 44.3% to the transformation efficiency of D-xylulose.
(4) extraction of D-xylulose is refining: first fermented liquid is adopted to the degerming of usual manner Plate Filtration, then to adopt molecular weight cut-off be the ultrafiltration membrance filter of 2000 Da, then carry out decolorizing with activated carbon and ion-exchange.Reconcentration to dry matter content is 90%, is then cooled to 48 ℃,, is more naturally cooled to room temperature and carries out crystallization as crystal seed to the D-xylulose of dry matter content 3% in concentrated solution, centrifugal after crystallization, obtains D-xylulose.
(5) enzymatic conversion: refining D-xylulose is made into 35% the aqueous solution, and regulating pH is 8.0, then adds magnesium sulfate and SO in the aqueous solution
2, the concentration to magnesium sulfate in the aqueous solution is 43 mg/L, SO
2concentration in the aqueous solution is 110 mg/L, and the D-xylulose aqueous solution is with 3 m
3/ h flow velocity flows through enzyme isomery post, and isomery post is containing 200 kilograms of xylose isomerases, and 58 ℃ of temperature of reaction must contain the isomery liquid glucose (containing D-xylulose 45.2% and D-wood sugar 53.4%, oligosaccharides 1.4%) of D-xylulose and D-wood sugar.
(6) wood sugar extracts refining: utilize the simulation moving-bed device of 12 post to process isomery liquid glucose, separate D-xylulose and D-wood sugar; Using calcium type Zeo-karb as sorbent material, take water as eluent, continuously feeding at 58 ℃, water inlet, discharging, obtain being rich in D-xylulose component (D-xylulose 92.0%, concentration 26.46%) and be rich in D-wood sugar (D-wood sugar 96.3%, concentration 29.32%) component; To be rich in wood sugar component and cross in a usual manner ion exchange resin, and remove impurity, being then concentrated into dry matter content is 90%, naturally be cooled to 60 ℃, to the wood sugar crystal seed that adds relative dry matter content 4% in concentrated solution, be naturally cooled to room temperature, centrifugal, obtain crystallization D-wood sugar; Being rich in xylulose component returns step (5) and carries out enzyme isomery.
(7) hydrogenation: crystalline xylose is made into 40% solution, adds thunder Buddhist nun ruthenium with solution weight 0.5%, at 140 ℃, under 7.6 Mpa hydrogen pressures, carry out hydrogenation reaction, reaction times 2 h, obtains xylitol solution.
(8) extraction of Xylitol is refining: xylitol solution is carried out in the usual way to activated carbon decolorizing, Plate Filtration and crosses ion exchange resin and remove impurity, being concentrated into dry matter content is 75%, then be naturally cooled to 50 ℃, in concentrated solution, add the Xylitol of dry matter content 2% as crystal seed, then be naturally cooled to room temperature and carry out crystallization, after crystallization, crystal is centrifugal, 55 ℃ of oven dry, obtain D-Xylitol.
embodiment 3
(1) substratum: seed culture medium A is: glucose 60 g/L, corn steep liquor 5 g/L, yeast extract paste 5 g/L, MgSO
41.5 g/L, natural pH; Seed culture medium B consists of: arabitol 100 g/L, yeast extract paste 5 g/L, calcium carbonate 0.5 g/L, pH 5.4; Fermention medium consists of: glucose 120 g/L, corn steep liquor 5 g/L, yeast extract paste 5 g/L, KH
2pO
44 g/L, MgSO
41.5 g/L, pH 5.5.
(2) seed liquor preparation: a) Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 seed liquor: get a ring bacterium from inclined-plane, be seeded in (culture medium A) in shaking flask, liquid amount 15%, 100 rpm, cultivate 20 h as yeast shaking flask kind liquid, then by 5% inoculum size, yeast shaking flask kind liquid are seeded in 100 L fermentor tank kind liquid (culture medium A) for 30 ℃, liquid amount 60%, ventilation ratio is 0.3 L/Lmin, 200 rpm, and 30 ℃ of enlarged culturing 20 h are as yeast kind of the liquid that spreads cultivation; B) gluconobacter oxydans seed liquor: the whole bacterium colonies of wash-out from a gluconobacter oxydans inclined-plane, be seeded in (substratum B) in shaking flask, liquid amount 15%, 180 rpm, cultivate 24 h as gluconobacter oxydans shaking flask kind liquid, then by 5% inoculum size, gluconobacter oxydans shaking flask kind liquid are seeded in 100 L fermentor tank kind liquid (substratum B) for 30 ℃, liquid amount 60%, ventilation ratio is 0.3 L/Lmin, 200 rpm, and 30 ℃ of enlarged culturing 20 h are as gluconobacter oxydans kind of the liquid that spreads cultivation.
(3) mixed fungus fermentation: by 5% inoculum size, kind of the liquid that first yeast spread cultivation is seeded in 1m
3in fermentor tank (fermention medium), liquid amount 60%, 100 rpm, ventilation ratio is 0.8 L/Lmin, 30 ℃ of fermentation culture 65 h, then by 15% inoculum size, kind of the liquid that again gluconobacter oxydans spread cultivation is seeded in fermented liquid relaying supervention ferment, in process, stream adds glucose and 10wt%KOH solution, ventilation ratio is 0.3 L/Lmin, 100 rpm, controlled fermentation liquid glucose content at 10 g/L and pH 5.0, 130 h ferment, stop mending sugar, treat that glucose and arabitol exhaust lower tank, in Liquid Detection fermented liquid, the content of D-xylulose is 136.3 g/L, glucose is 41.2% to the transformation efficiency of D-xylulose.
(4) extraction of D-xylulose is refining: first fermented liquid is adopted to the degerming of usual manner Plate Filtration, then to adopt molecular weight cut-off be the ultrafiltration membrance filter of 3000 Da, then carry out decolorizing with activated carbon and ion-exchange.Reconcentration to dry matter content is 95%, is then cooled to 55 ℃, in concentrated solution, adds the D-xylulose of dry matter content 4% as crystal seed, is more naturally cooled to room temperature and carries out crystallization, centrifugal after crystallization, obtains D-xylulose.
(5) enzymatic conversion: refining D-xylulose is made into 40% the aqueous solution, and regulating pH is 8.2, then adds magnesium sulfate and SO in the aqueous solution
2, the concentration to magnesium sulfate in the aqueous solution is 45 mg/L, SO
2concentration in the aqueous solution is 120 mg/L, and the D-xylulose aqueous solution is with 4 m
3/ h flow velocity flows through enzyme isomery post, and isomery post is containing 400 kilograms of xylose isomerases, and 55 ℃ of temperature of reaction must contain the isomery liquid glucose (containing D-xylulose 48.5% and D-wood sugar 50.1%, oligosaccharides 1.4%) of D-xylulose and D-wood sugar.
(6) wood sugar extracts refining: utilize the simulation moving-bed device of 12 post to process isomery liquid glucose, separate D-xylulose and D-wood sugar; Using calcium type Zeo-karb as sorbent material, take water as eluent, continuously feeding at 50 ℃, water inlet, discharging, obtain being rich in D-xylulose component (D-xylulose 89.3%, concentration 26.34%) and be rich in D-wood sugar (D-wood sugar 92.0%, concentration 28.33%) component; To be rich in wood sugar component and cross in a usual manner ion exchange resin, and remove impurity, being then concentrated into dry matter content is 75%, naturally be cooled to 40 ℃, to the wood sugar crystal seed that adds relative dry matter content 5% in concentrated solution, be naturally cooled to room temperature, centrifugal, obtain crystallization D-wood sugar; Being rich in xylulose component returns step (5) and carries out enzyme isomery.
(7) hydrogenation: crystalline xylose is made into 42% solution, adds Raney's nickel with solution weight 0.8%, at 170 ℃, under 7.2 Mpa hydrogen pressures, carry out hydrogenation reaction, reaction times 4 h, obtains xylitol solution.
(8) extraction of Xylitol is refining: xylitol solution is carried out in the usual way to activated carbon decolorizing, Plate Filtration and crosses ion exchange resin and remove impurity, being concentrated into dry matter content is 90%, then be naturally cooled to 60 ℃, in concentrated solution, add the Xylitol of dry matter content 3% as crystal seed, then be naturally cooled to room temperature and carry out crystallization, after crystallization, crystal is centrifugal, 75 ℃ of oven dry, obtain D-Xylitol.
comparative example
(1) substratum composition and kind liquid and preparation method thereof are with embodiment 1.
(2) mixed fungus fermentation: respectively by 10% inoculum size, yeast kind of liquid and gluconobacter oxydans kind of the liquid that spreads cultivation that spreads cultivation is seeded in to 1m simultaneously
3in fermentor tank (fermention medium), liquid amount 60%, 200 rpm, ventilation ratio is 0.5 L/Lmin, 35 ℃ of fermentation culture 85 h, treat that glucose drops to below 5 g/L, mend sugar, controlled fermentation liquid glucose content at 5 about g/L and pH 5.1,145 h that ferment, stop mending sugar, treat that glucose and arabitol exhaust lower tank, in Liquid Detection fermented liquid, the content of D-xylulose is 36.1 g/L, and glucose is 11.5 % to the transformation efficiency of D-xylulose.
(3) other step conditions are with embodiment 1.
Can find out from above-described embodiment and comparative example, the inventive method xylulose output and transformation efficiency improve greatly, are more beneficial to the industrial applications that biological fermentation is prepared Xylitol.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments, and the apparent variation of being extended out thus or variation are still among the protection domain in the invention.
Claims (9)
1. a microbial transformation glucose is prepared the method for D-xylulose, take glucose as raw material, by can transforming glucose the osmophilic yeast that is arabitol and can transform the gluconobacter oxydans that arabitol is D-xylulose (Gluconobacter oxydans) mixed fungus fermentation and cultivate to obtain D-xylulose fermented liquid, fermented liquid is through further processing to obtain D-xylulose, it is characterized in that, mixed fungus fermentation comprises the following steps: first, prepare respectively osmophilic yeast kind liquid and gluconobacter oxydans kind liquid, then first osmophilic yeast kind liquid is inoculated into containing carrying out fermentation culture in the fermention medium of glucose, be arabitol by conversion of glucose, mixed fungus fermentation in the osmophilic yeast fermentation middle and later periods is inoculated into fermented liquid by gluconobacter oxydans kind liquid again, in controlled fermentation liquid, glucose content is 5-10 g/L simultaneously, continue fermentation arabitol is converted into D-xylulose, must contain the fermented liquid of D-xylulose, describedly can transforming glucose be arabitol osmophilic yeast is Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 CGMCC NO.6197, the described arabitol that transforms is that the gluconobacter oxydans of D-xylulose is gluconobacter oxydans (Gluconobacter oxydans) NH-10 CGMCC No.2709.
2. microbial transformation glucose according to claim 1 is prepared the method for D-xylulose, it is characterized in that:
Osmophilic yeast kind liquid adopts following method preparation: get the osmophilic yeast bacterial classification that a transfering loop inclined-plane is preserved, be seeded in seed culture medium A, under 100-250 rpm, 30-35 ℃ condition, cultivate 10-20 h, obtain one-level kind liquid, gained one-level kind liquid is inoculated in seed culture medium A by the inoculum size of 5-15%, be enlarged culturing 12-20 h under 0.3-0.8 L/Lmin, 30-35 ℃ condition at 100-200 rpm, ventilation ratio, obtain osmophilic yeast kind liquid; Seed culture medium A consists of: glucose 60-80 g/L, corn steep liquor 5-10 g/L, yeast extract paste 3-5 g/L, MgSO
40.5-1.5 g/L, natural pH;
Gluconobacter oxydans kind liquid adopts following method preparation: the whole thalline on a gluconobacter oxydans inclined-plane are inoculated in seed culture medium B, under 100-250 rpm, 30-35 ℃ condition, cultivate 16-24 h, obtain one-level kind liquid, gained one-level kind liquid is inoculated in seed culture medium B by the inoculum size of 5-15%, be enlarged culturing 16-20 h under 0.3-0.8 L/Lmin, 30-35 ℃ condition at 100-200 rpm, ventilation ratio, obtain gluconobacter oxydans kind liquid; Seed culture medium B consists of: arabitol 100-120 g/L, yeast extract paste 5-10 g/L, calcium carbonate 0.5-1.5 g/L, pH 5.1-5.4.
3. microbial transformation glucose according to claim 1 is prepared the method for D-xylulose, it is characterized in that: mixed fungus fermentation specifically comprises the following steps: first, prepare respectively osmophilic yeast kind liquid and gluconobacter oxydans kind liquid, then press the inoculum size of 5-15%, first osmophilic yeast kind liquid is inoculated in the fermention medium that contains glucose sugar, being that 0.3-0.8 L/Lmin, 30-35 ℃ of condition bottom fermentation cultivated 65-85 h at 100-200 rpm, ventilation ratio, is arabitol by conversion of glucose; And then gluconobacter oxydans kind liquid is inoculated in fermented liquid by the inoculum size of 5-15%, be mixed fungus fermentation under 0.3-0.8 L/Lmin, 30-35 ℃ condition at 100-200 rpm, ventilation ratio; In fermenting process, stream adds glucose and alkaline substance solution, making glucose content in fermented liquid is that 5-10 g/L, pH are 4.6-5.4, whole fermentation time continues to stop supplementing glucose after 130-150 h, after glucose and arabitol exhaust, stop fermentation, must contain the fermented liquid of D-xylulose;
Described fermention medium consists of: glucose 120-160 g/L, corn steep liquor 5-10 g/L, yeast extract paste 3-5 g/L, KH
2pO
43-4 g/L, MgSO
40.5-1.5 g/L, pH 5.0-5.5.
4. microbial transformation glucose according to claim 1 is prepared the method for D-xylulose, it is characterized in that: the last handling process of D-xylulose fermented liquid is: first fermented liquid is adopted to the degerming of usual manner Plate Filtration, adopting molecular weight cut-off is the ultrafiltration membrance filter of 1000-3000 Da again, then carry out decolorizing with activated carbon and ion-exchange, fermented liquid being concentrated into dry matter content is 80-95% again, then be cooled to 40-55 ℃, in concentrated solution, add the D-xylulose of dry matter content 2-4% as crystal seed, naturally be cooled to again room temperature and carry out crystallization, centrifugal after crystallization, obtain D-xylulose.
5. a microbial transformation glucose is prepared the method for Xylitol, take glucose sugar as raw material, be D-xylulose by mixed fungus fermentation by conversion of glucose, by enzyme isomery, D-xylulose is converted into D-wood sugar again, then D-wood sugar hydrogenating reduction is Xylitol, it is characterized in that: the method that the microbial transformation glucose in employing claim 1-4 described in any one is prepared D-xylulose is D-xylulose by conversion of glucose.
6. method according to claim 5, is characterized in that: adopt xylose isomerase D-xylulose to be converted into the mixed solution of D-wood sugar and D-xylulose, then the separation of D-wood sugar, hydrogenation are obtained to Xylitol.
7. method according to claim 5, is characterized in that comprising the following steps:
(1) preparation of D-xylulose: the microbial transformation glucose described in employing claim 1 or 2 is prepared the method for D-xylulose and prepared D-xylulose;
(2) preparation of D-wood sugar: the D-xylulose in step (1) is made into the aqueous solution, makes the isomery liquid glucose that contains D-xylulose and D-wood sugar under the effect of xylose isomerase;
(3) extraction of D-wood sugar is refining: utilize the isomery liquid glucose in simulation moving-bed device treatment step (2), D-xylulose is separated with D-wood sugar and obtain being rich in D-xylulose component and be rich in D-wood sugar component, to be rich in D-wood sugar component crosses ion exchange resin and removes impurity, then concentrated, crystallization, obtains D-wood sugar; Being rich in D-xylulose component enters step (2) and applies mechanically;
(4) preparation of Xylitol: the D-wood sugar of step (3) is made into the aqueous solution, carries out hydrogenation reaction under catalyzer exists, obtain xylitol solution;
(5) extraction of Xylitol is refining: xylitol solution is removed by filter to catalyzer, crosses ion exchange resin and remove impurity, and then concentrated, crystallization, obtains D-Xylitol.
8. microbial transformation glucose according to claim 7 is prepared the method for Xylitol, it is characterized in that:
The preparation of D-wood sugar specifically comprises the following steps: the D-xylulose in step (1) is made into the aqueous solution of 35-45%, adjusting pH is 8.0-8.3, then adds magnesium sulfate and SO in the aqueous solution
2, the concentration to magnesium sulfate in the aqueous solution is 43-48 mg/L, SO
2concentration in the aqueous solution is 95-120 mg/L, then by the D-xylulose aqueous solution at 55-60 ℃, with 2-4 m
3the flow velocity of/h flows through the enzyme isomery post that is filled with xylose isomerase, must contain the isomery liquid glucose of D-xylulose and D-wood sugar;
The extraction of D-wood sugar is refined and is specifically comprised the following steps: using calcium type Zeo-karb as sorbent material, take water as eluent, at 50-65 ℃, utilize the isomery liquid glucose in simulation moving-bed device treatment step (2), obtain being rich in D-xylulose component and be rich in D-wood sugar component, to be rich in wood sugar component and carry out successively activated carbon decolorizing, Plate Filtration degerming and ion exchange resin removal of impurities, then being concentrated into dry matter content is 75-90%, then be cooled to 40-60 ℃, in concentrated solution, add the D-wood sugar of dry matter content 3-5% as crystal seed, naturally be cooled to again room temperature and carry out crystallization, centrifugal after crystallization, obtain D-wood sugar, being rich in D-xylulose component enters step (2) and applies mechanically,
The preparation of Xylitol specifically comprises the following steps: the aqueous solution that the D-wood sugar in step (3) is made into 40-50%, then add the Raney's nickel of aqueous solution weight 0.3-0.8% or thunder Buddhist nun ruthenium as catalyzer, at 140-170 ℃, 7.2-8.0 carry out hydrogenation reaction under MPa hydrogen pressure, reaction 2-4 h, obtains xylitol solution;
The extraction of Xylitol is refined and is specifically comprised the following steps: xylitol solution is carried out to activated carbon decolorizing, Plate Filtration degerming and ion exchange resin removal of impurities, then being concentrated into dry matter content is 75-90%, then be naturally cooled to 40-60 ℃, in concentrated solution, add the Xylitol of dry matter content 2-4% as crystal seed, then be naturally cooled to room temperature and carry out crystallization, after crystallization, crystal is centrifugal, 55-70 ℃ of oven dry, obtains D-Xylitol.
9. the osmophilic yeast bacterial classification that transforming glucose is arabitol, is characterized in that: described bacterial classification is Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 CGMCC NO.6197.
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