CN102796797A - 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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- CN102796797A CN102796797A CN2012102819672A CN201210281967A CN102796797A CN 102796797 A CN102796797 A CN 102796797A CN 2012102819672 A CN2012102819672 A CN 2012102819672A CN 201210281967 A CN201210281967 A CN 201210281967A CN 102796797 A CN102796797 A CN 102796797A
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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 midbody---the preparation method of D-xylulose, the method for utilizing this intermediate preparation Xylitol and used bacterial classification, being specifically related to a kind of is that raw material prepares the method for D-xylulose and Xylitol and used bacterial classification through microbial transformation with glucose.
Background technology
Xylitol belongs to five carbon polyols, is a kind of natural sweeteners.Xylitol is not utilized by bacterium in the oral cavity, has good preventing caries function.On the other hand, Xylitol need not Regular Insulin and participates in, and can directly get into human body cell and carry out the 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; Compare with other sugar alcohols, it has the highest solution heat, and edible have refrigerant sense, so Xylitol is a kind of ideal sucrose substitute, is widely used in chewing gum, toothpaste, sugarfree foods, medicine and other fields in recent years.
The working method of Xylitol can be divided into following two kinds at present:
1. chemical synthesis: be the method that production Xylitol commonly used is gone up in industry; Its ultimate principle is that pentosan obtains wood sugar through acid hydrolysis; Wood sugar hydrogenation under the katalysis of nickel generates Xylitol; Though this method raw material is easy to get; But its complex process, by product are many, separate purification difficult, consume a large amount of soda acids in, the process high to equipment requirements and produce a large amount of wastes, contaminate environment is serious, causes present Xylitol price higher, and this has also limited the widespread use of Xylitol to a certain extent.
2. biotransformation method: being divided into (1) according to the raw material difference is raw material with the wood sugar: with the xylan in agricultural wastes such as corn cob, straw, bagasse etc. behind dilute acid hydrolysis; Obtain the hydrolyzed solution that primary product is a wood sugar; Utilize the wood sugar in the microbial fermentation hydrolyzed solution to generate Xylitol then; But this method fermented liquid Xylitol content is very low, and the separation and Extraction cost is still very high, does not have the industriallization meaning; (2) with glucose be raw material: D-glucose changes into the D-arabitol under the effect of osmophilic yeast; Then the D-arabitol is oxidized to the D-xylulose under the effect of gluconobacter oxydans; Last D-xylulose generates Xylitol through the reductive action of yeast (Candida guilliermodii), is called three step fermentation methods again.But in this method the first step fermentation, the product arabitol has tangible product and suppresses phenomenon, causes the pectinose determining alcohol on the low side, and the 3rd step yeast conversion D-xylulose is that Xylitol efficient is lower in addition, and production concentration is no more than 30g/L.Therefore, bio-transformation is at present produced the problem that Xylitol mainly exists and is: conversion of glucose generates the pure and mild D-xylulose of pectinose and transforms and produce the Xylitol inefficiency.
Patent EP403392A and patent EP421882A disclose such method; Earlier produce arabitol with osmophilic yeast; Through the bacterium of adopting acetobacter, Gluconobacter, klebsiella arabitol is converted into the D-xylulose then; Transforming the D-xylulose through the effect of glucose (wood sugar) isomerase is the mixed solution of D-wood sugar and D-xylulose, above mixed solution hydrogenation obtain Xylitol.Though it is the problem of Xylitol inefficiency that this method has solved biological process conversion D-xylulose, osmophilic yeast generates the arabitol low efficiency problem and still exists, and makes this method production cost still be higher than chemical synthesis.
In the bio-transformation glucose production Xylitol technology, adopt the osmophilic yeast transforming glucose to produce arabitol, arabitol can produce thalline and suppress phenomenon, causes middle and later periods arabitol formation speed to reduce.The osmophilic yeast transforming glucose is arabitol ph optimum 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, and the about 20-30 h of 150 g/L arabitols can transform, ph optimum 5.0-5.5, and temperature is 30-35 ℃, needs yeast extract paste, MgSO
4, KH
2PO
4Deng as nutritive ingredient.From above growth and the bio-transformation condition basically identical that can find out osmophilic yeast and gluconobacter oxydans; Utilize this characteristic, attempt adopting the mikrobe mixed fungus fermentation to generate the method for Xylitol among the patent CN200410031949.4, this method is cultivated earlier yeast saccharomyces cerevisiae and gluconobacter oxydans kind liquid respectively; Then with 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 bigger, 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 efficient of producing Xylitol is low, does not have industrial value.
Summary of the invention
The present invention is directed to conversion of glucose in the prior art and become the defective of arabitol inefficiency; Provide a kind of microbial transformation glucose to prepare the method for D-xylulose; This method can be removed the feedback inhibition of arabitol, and the productive rate of final D-xylulose is effectively improved.
The method that the present invention also provides the D-xylulose that utilizes aforesaid method to make to prepare Xylitol, this method gained Xylitol productive rate significantly improves.
It is the osmophilic yeast bacterial classification of arabitol that the present invention also provides transforming glucose---Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 CGMCC NO.6197.
The contriver finds that through experimental study the efficient of osmophilic yeast transforming glucose in early stage generation arabitol is higher; Increase along with the pectinose determining alcohol; Conversion rate descends; 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 concn condition, 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 efficient.In research process; The contriver has attempted first cultivation 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 prepare the D-xylulose; Discovery adopts the mixed fungus fermentation of this mode can remove the feedback inhibition of arabitol, significantly improves the efficient that conversion of glucose is the D-xylulose.The contriver further studies, and has obtained producing the technical scheme of D-xylulose, and is as follows:
A kind of microbial transformation glucose prepares the method for D-xylulose; With D-glucose is raw material; But through with transforming glucose be arabitol osmophilic yeast with can transform the gluconobacter oxydans that arabitol is the D-xylulose (Gluconobacter oxydans) mixed fungus fermentation and must contain D-xylulose fermented liquid, fermented liquid through further handle the D-xylulose, it is characterized in that; Mixed fungus fermentation may further comprise the steps: at first; Preparing osmophilic yeast kind liquid and gluconobacter oxydans kind liquid respectively, earlier osmophilic yeast kind liquid is inoculated into then and carries out fermentation culture in the fermention medium that contains glucose, is arabitol with conversion of glucose; Again gluconobacter oxydans kind liquid is inoculated in the fermented liquid in the osmophilic yeast fermentation middle and later periods; Control simultaneously that glucose content is 5-10 g/L in the fermented liquid, continue fermentation arabitol is converted into the D-xylulose, must contain the fermented liquid of D-xylulose.
The main innovate point of above-mentioned preparation D-xylulose method be with the method for mixed fungus fermentation by original two kinds of bacteria culture fluids are added simultaneously contain carry out mixed fungus fermentation in the dextrose culture-medium; It is arabitol that the mode that makes conversion of glucose become the pure and mild arabitol of pectinose to be converted into the D-xylulose becomes elder generation's inoculation osmophilic yeast transforming glucose in glucose; Inoculating gluconobacter oxydans the middle and later periods in fermentation ferments arabitol is converted into the D-xylulose; In the control process about the concentration 10g/L of glucose; Remove the feedback inhibition of arabitol,, improve the productive rate of D-xylulose to improve the transformation efficiency of arabitol.
Fig. 2 produces Xylitol midbody (D-xylulose) canonical process graphic representation for the mikrobe mixed fungus fermentation; As can be seen from the figure: after the osmophilic yeast inoculation, OD value (biomass) increases sharply, and gets into stationary phase during 20 h; Meanwhile, arabitol begins to generate; At 20-80 h, thalli growth is slow, and glucose consumes fast, and arabitol generates fast; Behind 80 h inoculation gluconobacter oxydans, diauxic growth appears in thalline, and arabitol content descends rapidly; Drop to 3 g/L during 100 h, keep lower level ever since, D-xylulose content raises rapidly in the process; The OD value descends behind fermentation 140 h; D-xylulose throughput rate descends gradually, 150 h that ferment, D-xylulose content 148 g/L in the fermented liquid.
In the aforesaid method; Used osmophilic yeast and gluconobacter oxydans can be disclosedly in the prior art to can be used for all osmophilic yeast bacterial classifications and the gluconobacter oxydans bacterial classification that conversion of glucose is the D-xylulose; Disclosed bacterial classification among patent EP403392A, patent EP421882A and the patent CN200410031949.4 for example can play all that to remove the arabitol feedback inhibition, improve conversion of glucose be the purpose of D-xylulose efficient as long as carry out mixed fungus fermentation according to mode of the present invention.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 the aforesaid method; But being the osmophilic yeast of arabitol, transforming glucose preferentially select for use the contriver from the Ao Mo of row filter Kodak yeast (Kodamaea ohmeri) TL-1123; This TL-1123 bacterial classification is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preservation date is on June 6th, 2012, and deposit number is CGMCC NO.6197.This bacterial classification can anti-height oozes, high temperature resistant, can the normal growth breeding under 200 g/L glucose and 35 ℃ of conditions, efficient transforming glucose generation arabitol, transformation efficiency can reach 44%, the content of D-xylulose can reach 150 g/L in the fermented liquid.This bacterial screening process is:
1. with samples such as peach, pumpkin, Pulp Citrulli Pollens, (2 * 25 cm test tubes, liquid amount 10 mL) cultivate 2 ~ 4 d in 35 ° of C in the access enrichment medium under 220 r/min under aseptic condition.Enrichment medium is formed (gL
-1): glucose 50, yeast extract paste 3, peptone 3.
2. move with 10% inoculum size with liquid-transfering gun transfer enrichment medium that 10 mL are housed of the nutrient solution in is 1. shaken and cultivates 1 ~ 3 day in the pipe, so repeat 3 ~ 4 times, the dilution of bacterium liquid is coated carried out plate in the isolation medium and separate, cultivate 2 ~ 4 d down in 35 ° of C.Plate isolation and slant preservation substratum are formed (gL
-1): enrichment medium+agar 20.
3. will separate the single bacterium colony enlarged culturing that obtains; After 30 ° of C cultivate 2 ~ 4 d down; Insert respectively in the triangular flask that 50 mL primary dcreening operation fermentation cultures are housed (250 mL shake bottled 50 mL fermented liquids), 35 ° of C cultivate 3 d down, after fermentation culture finishes; Detect D-arabitol content, leave and take the higher bacterial strain of productive rate and carry out multiple sieve.The primary dcreening operation fermention medium is formed (gL
-1): glucose 50, yeast extract paste 3, peptone 3, CaCO
315.
4. will sieve bacterial strain again inserts and to be equipped with in the 250 mL triangular flasks that 50 mL sieve fermentation culture again; 35 ° of C cultivate 3 d down; After fermentation culture finishes, detect D-arabitol content, choose the high bacterial strain of arabitol productive rate and carry out further fermenting experiment and strain identification experiment.Sieve fermention medium again and form (gL
-1): glucose 200, yeast extract paste 3, peptone 3, CaCO
315.
Further, in the aforesaid method, can transform arabitol and be disclosed gluconobacter oxydans (Gluconobacter oxydans) NH-10 among the preferred patent CN200910024579.4 of gluconobacter oxydans of D-xylulose, preserving number: CGMCC No.2709.
Further, in the aforesaid method, mixed fungus fermentation specifically may further comprise the steps: at first; Prepare osmophilic yeast kind liquid and gluconobacter oxydans kind liquid respectively, the inoculum size of pressing 5-15% then is inoculated into osmophilic yeast kind liquid in the fermention medium that contains glucose sugar earlier; At 100-200 rpm; Ventilation is than being 0.3-0.8 L/Lmin, and 30-35 ℃ of condition bottom fermentation cultivated 65-85 h, is arabitol with conversion of glucose; And then the inoculum size of gluconobacter oxydans kind liquid by 5-15% be inoculated in the fermented liquid; At 100-200 rpm; Ventilation continues fermentation than being 0.3-0.8 L/Lmin under the 30-35 ℃ of condition, stream adds glucose and alkaline substance solution (alkaline substance solution is the aqueous solution of sodium hydroxide, Pottasium Hydroxide, yellow soda ash, salt of wormwood etc.) in fermented liquid simultaneously; Make that glucose content is that 5-10 g/L, pH are 4.6-5.4 in the fermented liquid; Whole fermentation time continues to stop to replenish glucose behind the 130-150 h, treats to stop after glucose and arabitol exhaust fermentation, must contain the fermented liquid of D-xylulose.
Further, fermention medium consists of: glucose 120-160 g/L, steeping water 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; The osmophilic yeast seed liquor adopts following method preparation: get the osmophilic yeast bacterial classification that a transfering loop inclined-plane is preserved, be seeded among the seed culture medium A, under 100-250 rpm, 30-35 ℃ condition, cultivate 10-20 h; Get one-level kind liquid; The inoculum size of gained one-level kind liquid by 5-15% is inoculated among the seed culture medium A, than being enlarged culturing 12-20 h under 0.3-0.8 L/Lmin, the 30-35 ℃ condition, gets osmophilic yeast kind liquid in 100-200 rpm, ventilation; Seed culture medium A consists of: glucose 60-80 g/L, steeping water 5-10 g/L, yeast extract paste 3-5 g/L, MgSO
40.5-1.5 g/L, natural pH;
The gluconobacter oxydans seed liquor adopts following method preparation: the whole thalline on the gluconobacter oxydans inclined-plane are inoculated among the seed culture medium B; Under 100-250 rpm, 30-35 ℃ condition, cultivate 16-24 h; Get one-level kind liquid; The inoculum size of gained one-level kind liquid by 5-15% is inoculated among the seed culture medium B, than being enlarged culturing 16-20 h under 0.3-0.8 L/Lmin, the 30-35 ℃ condition, gets gluconobacter oxydans kind liquid in 100-200 rpm, ventilation; Seed culture medium B consists of: arabitol 100-120 g/L, yeast extract paste 5-10 g/L, lime carbonate 0.5-1.5 g/L, pH 5.1-5.4.
Further; In the aforesaid method; The last handling process that contains D-xylulose fermented liquid is: fermented liquid is removed thalline and impurity through Plate Filtration, ultra-filtration membrane ultrafiltration, activated carbon decolorizing and IX; Concentrated then, crystallization gets the D-xylulose, is more specifically: fermented liquid is adopted the Plate Filtration degerming, uses the ultrafiltration membrance filter of molecular weight cut-off as 1000-3000 Da again, use decolorizing with activated carbon and ion exchange treatment then.Again the fermented liquid after the IX being concentrated into solids content is 80-95%; Be cooled to 40-55 ℃ then, the D-xylulose that in liquid concentrator, adds solids content 2-4% is cooled to room temperature more naturally and carries out crystallization as crystal seed; Centrifugal after the crystallization, obtain the D-xylulose.
The above-mentioned D-of making xylulose is the midbody of Xylitol preparation; Can further prepare Xylitol with the D-xylulose that aforesaid method makes; Step is: with glucose sugar is raw material; The D-xylulose that method through above-mentioned mixed fungus fermentation makes further adopts prior art to be reduced to Xylitol, promptly gets.
The technology that the D-xylulose is reduced to Xylitol has a lot; For example can adopt the reductive action of yeast (Candida guilliermodii) that the D-xylulose is reduced to Xylitol; Also can adopt xylose isomerase (also to be glucose isomerase; Transforming the D-xylulose earlier down together) is the mixed solution of D-wood sugar and D-xylulose, then the separation of D-wood sugar, hydrogenation is obtained Xylitol.Above-mentioned prior art all can be used among the present invention the D-xylulose being reduced to Xylitol, but the preferred xylose isomerase enzymatic conversion D-xylulose that adopts generates the D-wood sugar, adopts xylose isomerase gained Xylitol efficient higher, is beneficial to industrialized production more.
The method that the present invention prepares Xylitol preferably adopts mixed fungus fermentation to prepare the D-xylulose and adopts the two bonded mode of xylose isomerase enzymatic conversion D-xylulose to carry out, and gained Xylitol productive rate improves greatly, and the preferred method scheme is following:
A kind of microbial transformation glucose prepares the method for Xylitol; With glucose sugar is raw material; The method that adopts the mentioned microorganism transforming glucose to prepare the D-xylulose is the D-xylulose with conversion of glucose; Adopt xylose isomerase that the D-xylulose is converted into the mixed solution of D-wood sugar and D-xylulose, then the separation of D-wood sugar, hydrogenation obtained Xylitol, may further comprise the steps:
1. the preparation of D-xylulose: the method that adopts the mixed bacterium culture transformation glucose of mentioned microorganism to prepare the D-xylulose prepares the D-xylulose;
2. the preparation of D-wood sugar: the D-xylulose of step in 1. is made into the aqueous solution, under the effect of xylose isomerase, makes the isomery liquid glucose that contains D-xylulose and D-wood sugar;
3. the extraction of D-wood sugar is refining: utilize the isomery liquid glucose of simulation moving-bed apparatus processes step in 2.; The D-xylulose separated with the D-wood sugar 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; Concentrated then, crystallization get the D-wood sugar; Being rich in D-xylulose component gets into step and applies mechanically in 2.;
4. the preparation of Xylitol: the D-wood sugar of step in 3. is made into the aqueous solution, in the presence of catalyzer, carries out hydrogenation reaction, obtain xylitol solution;
5. the extraction of Xylitol is refining: the Xylitol reacting liquid filtering is removed catalyzer, is crossed ion exchange resin and remove impurity, concentrate then, crystallization, the D-Xylitol.
Further, the preparation of D-wood sugar specifically may further comprise the steps: the D-xylulose of step in 1. is made into the aqueous solution of 35-45%, and adjusting pH is 8.0-8.3, in the aqueous solution, adds sal epsom and SO again
2, to the concentration of sal epsom in the aqueous solution be 43-48 mg/L, SO
2Concentration in the aqueous solution is 95-120 mg/L, then with the D-xylulose aqueous solution with 2-4 m
3/ h flow velocity flows through enzyme isomery post, and the isomery post contains xylose isomerase 200-400 kilogram, 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 refining specifically may further comprise the steps: as sorbent material, is eluent with water with calcium type Zeo-karb; Under 50-65 ℃, utilize the isomery liquid glucose of simulation moving-bed apparatus processes step in 2., obtain being rich in D-xylulose component and be rich in D-wood sugar component, will be rich in the wood sugar component and carry out activated carbon decolorizing, Plate Filtration by usual manner and cross ion exchange resin and remove impurity; Being concentrated into solids content is more than the 75-90%; Naturally be cooled to 40-60 ℃ then, the D-wood sugar that in liquid concentrator, adds solids content 3-5% is cooled to room temperature more naturally and carries out crystallization as crystal seed; After the crystallization that crystal is centrifugal, obtain the D-wood sugar; Being rich in D-xylulose component gets into step and applies mechanically in 2..The ion exchange resin that uses when removing impurity has cationic ion exchange resin and anionic ion exchange resin; Wherein, The preferred large porous strong acid vinylbenzene of cationic ion exchange resin-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 may further comprise the steps: the aqueous solution that the D-wood sugar of step in 3. is made into 40-50%; The Raney's nickel of adding aqueous solution weight 0.3-0.8% or thunder Buddhist nun ruthenium at 140-170 ℃, carry out hydrogenation reaction under the 7.2-8.0 Mpa hydrogen pressure as catalyzer; Reaction 2-4 h obtains xylitol solution.
Further, the extraction of Xylitol is refining specifically to be may further comprise the steps: xylitol solution is carried out activated carbon decolorizing, Plate Filtration and mistake ion exchange resin by usual manner remove impurity, being concentrated into solids content is more than the 75-90%; Naturally be cooled to 40-60 ℃ then; The Xylitol that in liquid concentrator, adds solids content 2-4% is cooled to room temperature then and carries out crystallization as crystal seed, and is after the crystallization that crystal is centrifugal; 55-75 ℃ of oven dry gets the D-Xylitol.The ion exchange resin that uses when removing impurity has cationic ion exchange resin and anionic ion exchange resin; Wherein, The preferred large porous strong acid vinylbenzene of cationic ion exchange resin-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:
The mixed fungus fermentation technology that 1, will prepare the D-xylulose is improved; The inoculation osmophilic yeast carries out the arabitol fermentation earlier; Inoculate gluconobacter oxydans in the fermentation middle and later periods then; Removed the feedback inhibition of arabitol,, improved the productive rate of D-xylulose than the method transformation efficiency height of inoculating osmophilic yeast and gluconobacter oxydans in the prior art simultaneously; One-step fermentation can be the D-xylulose with conversion of glucose, and than earlier preparing arabitol with osmophilic yeast in the prior art, it is simple to operate to prepare the two-step fermenting of D-xylulose with arabitol again, and the cycle is short, has practiced thrift raw material, has reduced energy consumption.
2, preferably use as osmophilic yeast from the Ao Mo of row filter Kodak yeast (Kodamaea ohmeri) TL-1123; The anti-height of this bacterial strain oozes, high temperature resistant; Normal growth breeding under 200 g/L glucose and 35 ℃ of conditions; Efficiently transforming glucose generates arabitol, and transformation efficiency can reach 44%, and the content of D-xylulose can reach 150 g/L in the fermented liquid.
3, the D-xylulose that utilizes the mixed fungus fermentation method to make prepares Xylitol and has improved production efficiency, has reduced cost.Further, preferably adopt xylose isomerase to prepare the method for Xylitol, efficient further improves, and in actual production, is easier to operation and realization.
Description of drawings
For content of the present invention is more clearly understood, below according to a particular embodiment of the invention and combine accompanying drawing, the present invention is done further detailed explanation, wherein:
Fig. 1 prepares Xylitol and midbody D-xylulose process flow sheet thereof for microbial transformation glucose of the present invention;
Fig. 2 produces Xylitol midbody (D-xylulose) canonical process graphic representation for the mikrobe mixed fungus fermentation.
Preservation information
The used osmophilic yeast Ao Mo of the present invention Kodak yeast (Kodamaea ohmeri) TL-1123 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, and preservation date is on June 6th, 2012, and deposit number is CGMCC NO.6197.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete processing condition of embodiment, material proportion and result thereof only are used to explain the present invention, and should also can not limit the present invention described in claims.
Among the following embodiment, used osmophilic yeast is Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123, deposit number: CGMCC NO.6197; Used gluconobacter oxydans is gluconobacter oxydans (Gluconobacter oxydans) NH-10, deposit number: CGMCC NO.2079, and open in patent CN200910024579.4.Therefore, the used bacterial classification of the present invention satisfies and to disclose sufficient requirement.Ao Mo Kodak yeast and gluconobacter oxydans are preserved under 4 ℃ with the inclined-plane, and Ao Mo Kodak yeast preservation slant medium is formed (g/L): glucose 50, yeast extract paste 3, peptone 3, agar 20, natural pH; Gluconobacter oxydans preservation slant medium is formed (g/L): arabitol 80, and yeast extract paste 5, peptone 3, lime carbonate 1, agar 20, pH 5.2.
Among the following embodiment; Removing the used Zeo-karb of impurity is the mixture of Amberlite IRC50 and Amberlite FPC22Na; Anionite-exchange resin is the mixture of Amberlite FPA51 and Amberlite FPA90CI; Xylose isomerase is also referred to as glucose isomerase, adopts sugar alcohol industry fixed glucose isomerase commonly used.
Embodiment 1
(1) substratum: seed culture medium A consists of: glucose 80 g/L, steeping water 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, and yeast extract paste 10 g/L, lime carbonate 1.5 g/L, pH 5.2; Fermention medium consists of: glucose 160 g/L, steeping water 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 the inclined-plane, be seeded in and shake (culture medium A) in the bottle, liquid amount 15%; 250 rpm cultivate 16 h for 35 ℃ and shake bottle kind liquid (being primary seed solution, down together) as yeast; By 10% inoculum size yeast being shaken bottle kind liquid then is seeded in the 100 L fermentor tank kind liquid (culture medium A); Liquid amount 60%, ventilation be than being 0.5 L/Lmin, 150 rpm; 35 ℃ of enlarged culturing 14 h are as yeast kind of the liquid (be the osmophilic yeast seed liquor, down with) that spreads cultivation; B) gluconobacter oxydans seed liquor: the whole bacterium colonies of wash-out from the gluconobacter oxydans inclined-plane are seeded in and shake (substratum B) in the bottle, liquid amount 15%; 250 rpm cultivate 16 h for 35 ℃ and shake bottle kind liquid (being primary seed solution, down together) as gluconobacter oxydans; By 10% inoculum size gluconobacter oxydans being shaken bottle kind liquid then is seeded in the 100 L fermentor tank kind liquid (substratum B); Liquid amount 60%, ventilation be than being 0.5 L/Lmin, 200 rpm; 35 ℃ of enlarged culturing 16 h are as gluconobacter oxydans kind of the liquid (be the gluconobacter oxydans seed liquor, down with) that spreads cultivation.
(3) mixed fungus fermentation: by 10% inoculum size, earlier yeast kind of the liquid that spreads cultivation is seeded in 1m
3In the fermentor tank (fermention medium), liquid amount 60%, 200 rpm; Ventilation is than being 0.5 L/Lmin, and 35 ℃ of fermentation culture 85 h are then by 10% inoculum size; Again gluconobacter oxydans kind of the liquid that spreads cultivation is seeded in and continues mixed fungus fermentation in the fermented liquid, stream adds glucose and 10wt%Na in the process
2CO
3Solution, 150 rpm, ventilation is than being 0.5 L/Lmin; Glucose content is at 5 g/L in the control fermented liquid, and pH is 5.1, and (yeast fermentation+gluconobacter oxydans fermentation is 145h altogether for 145 h that ferment; Down together), stop to mend sugar, treat that glucose and arabitol exhaust following jar; The content of D-xylulose is 148.3 g/L in the Liquid Detection fermented liquid, and glucose is 43.2% to the transformation efficiency of D-xylulose.
(4) extraction of D-xylulose is refining: fermented liquid is at first adopted the degerming of usual manner Plate Filtration, and adopting molecular weight cut-off again is the ultrafiltration membrance filter of 1000 Da, carries out decolorizing with activated carbon and IX then.Reconcentration to solids content is 80%, is cooled to 40 ℃ then, and the D-xylulose that in liquid concentrator, adds solids content 2% is cooled to room temperature more naturally and carries out crystallization as crystal seed, and is centrifugal after the crystallization, obtains the D-xylulose.
(5) enzymatic conversion: will make with extra care the aqueous solution that the D-xylulose is made into 35-45%, adjusting pH is 8.0-8.3, in the aqueous solution, adds sal epsom and SO again
2, to the concentration of sal epsom in the aqueous solution be 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 the isomery post contains 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 apparatus processes isomery of 12 posts liquid glucose, separate D-xylulose and D-wood sugar; With calcium type Zeo-karb as sorbent material; With water is eluent, in 65 ℃ of following continuously feedings, water inlet, discharging, obtains 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 the wood sugar component and cross ion exchange resin in a usual manner, and remove impurity, being concentrated into solids content then is 80%; Naturally be cooled to 50 ℃, in liquid concentrator, add the wood sugar crystal seed of relative solids content 3%, be cooled to room temperature naturally; Centrifugal, obtain crystallization D-wood sugar; Being rich in the xylulose component returns step (5) and carries out the enzyme isomery.
(7) hydrogenation: crystalline xylose is made into 45% solution, adds Raney's nickel with solution weight 0.3 %, at 150 ℃, under the 8.0 Mpa hydrogen pressures, carry out hydrogenation reaction, reaction times 3 h obtains xylitol solution.
(8) extraction of Xylitol is refining: xylitol solution is carried out activated carbon decolorizing, Plate Filtration and crosses ion exchange resin removal impurity by usual manner; Being concentrated into solids content is 84%, is cooled to 40 ℃ then naturally, and the Xylitol that in liquid concentrator, adds relative solids content 4% is as crystal seed; Naturally be cooled to room temperature then and carry out crystallization; After the crystallization that crystal is centrifugal, 65 ℃ of oven dry get the D-Xylitol.
Embodiment 2
(1) substratum: seed culture medium A consists of: glucose 70 g/L, steeping water 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, and yeast extract paste 7 g/L, lime carbonate 1.2 g/L, pH 5.1; Fermention medium consists of: glucose 150 g/L, steeping water 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 the inclined-plane, be seeded in and shake (culture medium A) in the bottle, liquid amount 15%; 180 rpm cultivate 18 h for 33 ℃ and shake bottle kind liquid as yeast, by 15% inoculum size yeast are shaken bottle kind liquid then and are seeded in the 100 L fermentor tank kind liquid (culture medium A); Liquid amount 60%; Ventilation is than being 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 the gluconobacter oxydans inclined-plane are seeded in and shake (substratum B) in the bottle, liquid amount 15%; 100 rpm cultivate 20 h for 33 ℃ and shake bottle kind liquid as gluconobacter oxydans, by 15% inoculum size gluconobacter oxydans are shaken bottle kind liquid then and are seeded in the 100 L fermentor tank kind liquid (substratum B); Liquid amount 60%; Ventilation is than being 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, earlier yeast kind of the liquid that spreads cultivation is seeded in 1m
3In the fermentor tank (fermention medium), liquid amount 60%, 200 rpm, ventilation is than being 0.3 L/Lmin; 33 ℃ of fermentation culture 75 h then by 5% inoculum size, are seeded in fermented liquid relaying supervention ferment with gluconobacter oxydans kind of the liquid that spreads cultivation again, and ventilation is than being 0.8 L/Lmin; 100 rpm, stream adds glucose and 10wt%NaOH solution in the process, and glucose content is at 8 g/L in the control fermented liquid; PH is 4.6, and 150 h that ferment stop to mend sugar; Treat that glucose and arabitol exhaust following jar, the content of D-xylulose is 153.2 g/L in the Liquid Detection fermented liquid, and glucose is 44.3% to the transformation efficiency of D-xylulose.
(4) extraction of D-xylulose is refining: fermented liquid is at first adopted the degerming of usual manner Plate Filtration, and adopting molecular weight cut-off again is the ultrafiltration membrance filter of 2000 Da, carries out decolorizing with activated carbon and IX then.Reconcentration to solids content is 90%, is cooled to 48 ℃ then, and the D-xylulose of solids content 3% is cooled to room temperature more naturally and carries out crystallization as crystal seed in liquid concentrator, and is centrifugal after the crystallization, obtains the D-xylulose.
(5) enzymatic conversion: will make with extra care the D-xylulose and be made into 35% the aqueous solution, regulating pH is 8.0, in the aqueous solution, adds sal epsom and SO again
2, to the concentration of sal epsom in the aqueous solution be 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 the isomery post contains 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 apparatus processes isomery of 12 posts liquid glucose, separate D-xylulose and D-wood sugar; With calcium type Zeo-karb as sorbent material; With water is eluent, in 58 ℃ of following continuously feedings, water inlet, discharging, obtains 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 the wood sugar component and cross ion exchange resin in a usual manner, and remove impurity, being concentrated into solids content then is 90%; Naturally be cooled to 60 ℃, in liquid concentrator, add the wood sugar crystal seed of relative solids content 4%, be cooled to room temperature naturally; Centrifugal, obtain crystallization D-wood sugar; Being rich in the xylulose component returns step (5) and carries out the enzyme isomery.
(7) hydrogenation: crystalline xylose is made into 40% solution, adds thunder Buddhist nun ruthenium with solution weight 0.5%, at 140 ℃, under the 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 activated carbon decolorizing, Plate Filtration and crosses ion exchange resin removal impurity by usual manner; Being concentrated into solids content is 75%, is cooled to 50 ℃ then naturally, and the Xylitol that in liquid concentrator, adds solids content 2% is as crystal seed; Naturally be cooled to room temperature then and carry out crystallization; After the crystallization that crystal is centrifugal, 55 ℃ of oven dry get the D-Xylitol.
Embodiment 3
(1) substratum: seed culture medium A is: glucose 60 g/L, steeping water 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, and yeast extract paste 5 g/L, lime carbonate 0.5 g/L, pH 5.4; Fermention medium consists of: glucose 120 g/L, steeping water 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 the inclined-plane, be seeded in and shake (culture medium A) in the bottle, liquid amount 15%; 100 rpm cultivate 20 h for 30 ℃ and shake bottle kind liquid as yeast, by 5% inoculum size yeast are shaken bottle kind liquid then and are seeded in the 100 L fermentor tank kind liquid (culture medium A); Liquid amount 60%; Ventilation is than being 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 the gluconobacter oxydans inclined-plane are seeded in and shake (substratum B) in the bottle, liquid amount 15%; 180 rpm cultivate 24 h for 30 ℃ and shake bottle kind liquid as gluconobacter oxydans, by 5% inoculum size gluconobacter oxydans are shaken bottle kind liquid then and are seeded in the 100 L fermentor tank kind liquid (substratum B); Liquid amount 60%; Ventilation is than being 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, earlier yeast kind of the liquid that spreads cultivation is seeded in 1m
3In the fermentor tank (fermention medium), liquid amount 60%, 100 rpm, ventilation is than being 0.8 L/Lmin; 30 ℃ of fermentation culture 65 h then by 15% inoculum size, are seeded in fermented liquid relaying supervention ferment with gluconobacter oxydans kind of the liquid that spreads cultivation again; Stream adds glucose and 10wt%KOH solution in the process, ventilates than being 0.3 L/Lmin 100 rpm; 5.0,130 h that ferment stop to mend sugared control fermented liquid glucose content at 10 g/L and pH; Treat that glucose and arabitol exhaust following jar, the content of D-xylulose is 136.3 g/L in the Liquid Detection fermented liquid, and glucose is 41.2% to the transformation efficiency of D-xylulose.
(4) extraction of D-xylulose is refining: fermented liquid is at first adopted the degerming of usual manner Plate Filtration, and adopting molecular weight cut-off again is the ultrafiltration membrance filter of 3000 Da, carries out decolorizing with activated carbon and IX then.Reconcentration to solids content is 95%, is cooled to 55 ℃ then, and the D-xylulose that in liquid concentrator, adds solids content 4% is cooled to room temperature more naturally and carries out crystallization as crystal seed, and is centrifugal after the crystallization, obtains the D-xylulose.
(5) enzymatic conversion: will make with extra care the D-xylulose and be made into 40% the aqueous solution, regulating pH is 8.2, in the aqueous solution, adds sal epsom and SO again
2, to the concentration of sal epsom in the aqueous solution be 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 the isomery post contains 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 apparatus processes isomery of 12 posts liquid glucose, separate D-xylulose and D-wood sugar; With calcium type Zeo-karb as sorbent material; With water is eluent, in 50 ℃ of following continuously feedings, water inlet, discharging, obtains 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 the wood sugar component and cross ion exchange resin in a usual manner, and remove impurity, being concentrated into solids content then is 75%; Naturally be cooled to 40 ℃, in liquid concentrator, add the wood sugar crystal seed of relative solids content 5%, be cooled to room temperature naturally; Centrifugal, obtain crystallization D-wood sugar; Being rich in the xylulose component returns step (5) and carries out the enzyme isomery.
(7) hydrogenation: crystalline xylose is made into 42% solution, adds Raney's nickel with solution weight 0.8%, at 170 ℃, under the 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 activated carbon decolorizing, Plate Filtration and crosses ion exchange resin removal impurity by usual manner; Being concentrated into solids content is 90%, is cooled to 60 ℃ then naturally, and the Xylitol that in liquid concentrator, adds solids content 3% is as crystal seed; Naturally be cooled to room temperature then and carry out crystallization; After the crystallization that crystal is centrifugal, 75 ℃ of oven dry get the D-Xylitol.
Comparative example
(1) substratum is formed and is planted liquid and preparation method thereof with embodiment 1.
(2) mixed fungus fermentation:, yeast kind of liquid and gluconobacter oxydans kind of the liquid that spreads cultivation that spreads cultivation is seeded in 1m simultaneously respectively by 10% inoculum size
3In the fermentor tank (fermention medium), liquid amount 60%, 200 rpm, ventilation is than being 0.5 L/Lmin; 35 ℃ of fermentation culture 85 h treat that glucose drops to below 5 g/L, mend sugar; Control fermented liquid glucose content about 5 g/L with pH 5.1,145 h that ferment stop to mend sugar; Treat that glucose and arabitol exhaust following jar, the content of D-xylulose is 36.1 g/L in the Liquid Detection fermented liquid, and glucose is 11.5 % to the transformation efficiency of D-xylulose.
(3) other step conditions are with embodiment 1.
Can find out that from the foregoing description and comparative example the inventive method xylulose output and transformation efficiency improve greatly, be beneficial to the industrial applications that biological fermentation prepares Xylitol more.
Obviously, the foregoing description only be for explanation clearly done for example, and be not qualification to embodiment.For the those of ordinary skill in affiliated field, on the basis of above-mentioned explanation, can also make other multi-form variation or change.Here need not also can't give to all embodiments exhaustive, and the conspicuous variation of being extended out thus or the change still be among the protection domain of the invention.
Claims (10)
1. a microbial transformation glucose prepares the method for D-xylulose; With glucose is raw material; But through transforming glucose be arabitol osmophilic yeast with can transform the gluconobacter oxydans that arabitol is the D-xylulose (Gluconobacter oxydans) mixed fungus fermentation cultivate D-xylulose fermented liquid, fermented liquid through further handle the D-xylulose, it is characterized in that; Mixed fungus fermentation may further comprise the steps: at first; Preparing osmophilic yeast kind liquid and gluconobacter oxydans kind liquid respectively, earlier osmophilic yeast kind liquid is inoculated into then and carries out fermentation culture in the fermention medium that contains glucose, is arabitol with conversion of glucose; Again gluconobacter oxydans kind liquid is inoculated into mixed fungus fermentation in the fermented liquid in the osmophilic yeast fermentation middle and later periods; Control simultaneously that glucose content is 5-10 g/L in the fermented liquid, continue fermentation arabitol is converted into the D-xylulose, must contain the fermented liquid of D-xylulose.
2. microbial transformation glucose according to claim 1 prepares the method for D-xylulose, it is characterized in that: but said transforming glucose is the osmophilic yeast of arabitol is Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 CGMCC NO.6197; The said arabitol that transforms is that the gluconobacter oxydans of D-xylulose is gluconobacter oxydans (Gluconobacter oxydans) NH-10 CGMCC No.2709.
3. microbial transformation glucose according to claim 1 and 2 prepares 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 among the seed culture medium A; Under 100-250 rpm, 30-35 ℃ condition, cultivate 10-20 h, get one-level kind liquid, the inoculum size of gained one-level kind liquid by 5-15% is inoculated among the seed culture medium A; Than being enlarged culturing 12-20 h under 0.3-0.8 L/Lmin, the 30-35 ℃ condition, get osmophilic yeast kind liquid in 100-200 rpm, ventilation; Seed culture medium A consists of: glucose 60-80 g/L, steeping water 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 the gluconobacter oxydans inclined-plane are inoculated among the seed culture medium B; Under 100-250 rpm, 30-35 ℃ condition, cultivate 16-24 h; Get one-level kind liquid; The inoculum size of gained one-level kind liquid by 5-15% is inoculated among the seed culture medium B, than being enlarged culturing 16-20 h under 0.3-0.8 L/Lmin, the 30-35 ℃ condition, gets gluconobacter oxydans kind liquid in 100-200 rpm, ventilation; Seed culture medium B consists of: arabitol 100-120 g/L, yeast extract paste 5-10 g/L, lime carbonate 0.5-1.5 g/L, pH 5.1-5.4.
4. microbial transformation glucose according to claim 1 and 2 prepares the method for D-xylulose; It is characterized in that: mixed fungus fermentation specifically may further comprise the steps: at first; Prepare osmophilic yeast kind liquid and gluconobacter oxydans kind liquid respectively; Press the inoculum size of 5-15% then; Earlier osmophilic yeast kind liquid being inoculated in the fermention medium that contains glucose sugar, than cultivating 65-85 h for 0.3-0.8 L/Lmin, 30-35 ℃ condition bottom fermentation, is arabitol with conversion of glucose in 100-200 rpm, ventilation; And then the inoculum size of gluconobacter oxydans kind liquid by 5-15% be inoculated in the fermented liquid, at 100-200 rpm, ventilation than mixed fungus fermentation under for 0.3-0.8 L/Lmin, 30-35 ℃ condition; Stream adds glucose and alkaline substance solution in the fermenting process; Make that glucose content is that 5-10 g/L, pH are 4.6-5.4 in the fermented liquid; Whole fermentation time continues to stop to replenish glucose behind the 130-150 h; Treat to stop after glucose and arabitol exhaust fermentation, must contain the fermented liquid of D-xylulose;
Said fermention medium consists of: glucose 120-160 g/L, steeping water 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.
5. microbial transformation glucose according to claim 1 and 2 prepares the method for D-xylulose; It is characterized in that: the last handling process of D-xylulose fermented liquid is: fermented liquid is at first adopted the degerming of usual manner Plate Filtration, and adopting molecular weight cut-off again is the ultrafiltration membrance filter of 1000-3000 Da, carries out decolorizing with activated carbon and IX then; Again fermented liquid being concentrated into solids content is 80-95%; Be cooled to 40-55 ℃ then, the D-xylulose that in liquid concentrator, adds solids content 2-4% is cooled to room temperature more naturally and carries out crystallization as crystal seed; Centrifugal after the crystallization, obtain the D-xylulose.
6. a microbial transformation glucose prepares the method for Xylitol; With glucose sugar is raw material; Is the D-xylulose through mixed fungus fermentation with conversion of glucose; Through the enzyme isomery D-xylulose is converted into the D-wood sugar again, D-wood sugar hydrogenating reduction is an Xylitol then, it is characterized in that: the method that adopts each described microbial transformation glucose among the claim 1-6 to prepare the D-xylulose is the D-xylulose with conversion of glucose.
7. method according to claim 6 is characterized in that: adopt xylose isomerase that the D-xylulose is converted into the mixed solution of D-wood sugar and D-xylulose, then the separation of D-wood sugar, hydrogenation are obtained Xylitol.
8. according to claim 6 or 7 described methods, it is characterized in that may further comprise the steps:
(1) preparation of D-xylulose: the method that adopts claim 1 or 2 described microbial transformation glucose to prepare the D-xylulose prepares the D-xylulose;
(2) preparation of D-wood sugar: the D-xylulose in the step (1) is made into the aqueous solution, under the effect of xylose isomerase, makes the isomery liquid glucose that contains D-xylulose and D-wood sugar;
(3) extraction of D-wood sugar is refining: utilize the isomery liquid glucose in the simulation moving-bed apparatus processes step (2); The D-xylulose separated with the D-wood sugar 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; Concentrated then, crystallization get the D-wood sugar; Being rich in D-xylulose component entering step (2) applies mechanically;
(4) preparation of Xylitol: the D-wood sugar of step (3) is made into the aqueous solution, in the presence of catalyzer, carries out hydrogenation reaction, obtain xylitol solution;
(5) extraction of Xylitol is refining: xylitol solution is removed by filter catalyzer, crosses ion exchange resin and remove impurity, concentrate then, crystallization, the D-Xylitol.
9. microbial transformation glucose according to claim 8 prepares the method for Xylitol, it is characterized in that:
The preparation of D-wood sugar specifically may further comprise the steps: the D-xylulose in the step (1) is made into the aqueous solution of 35-45%, and adjusting pH is 8.0-8.3, in the aqueous solution, adds sal epsom and SO again
2, to the concentration of sal epsom in the aqueous solution be 43-48 mg/L, SO
2Concentration in the aqueous solution is 95-120 mg/L, then with the D-xylulose aqueous solution under 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 refining specifically to be may further comprise the steps: as sorbent material, is eluent with water with calcium type Zeo-karb, under 50-65 ℃, utilizes the isomery liquid glucose in the simulation moving-bed apparatus processes step (2); Obtain being rich in D-xylulose component and be rich in D-wood sugar component; To be rich in the wood sugar component and carry out activated carbon decolorizing, Plate Filtration degerming and ion exchange resin removal of impurities successively, then it being concentrated into solids content is 75-90%, is cooled to 40-60 ℃ then; The D-wood sugar that in liquid concentrator, adds solids content 3-5% is as crystal seed; Naturally be cooled to room temperature again and carry out crystallization, centrifugal after the crystallization, obtain the D-wood sugar; Being rich in D-xylulose component entering step (2) applies mechanically;
The preparation of Xylitol specifically may further comprise the steps: the aqueous solution that the D-wood sugar in the step (3) is made into 40-50%; The Raney's nickel or the thunder Buddhist nun ruthenium that add aqueous solution weight 0.3-0.8% then are as catalyzer; At 140-170 ℃; 7.2-8.0 carry out hydrogenation reaction under the MPa hydrogen pressure, reaction 2-4 h obtains xylitol solution;
The extraction of Xylitol is refining specifically to be may further comprise the steps: xylitol solution is carried out activated carbon decolorizing, Plate Filtration degerming and ion exchange resin removal of impurities; Then it being concentrated into solids content is 75-90%, is cooled to 40-60 ℃ then naturally, and the Xylitol that in liquid concentrator, adds solids content 2-4% is as crystal seed; Naturally be cooled to room temperature then and carry out crystallization; After the crystallization that crystal is centrifugal, 55-70 ℃ of oven dry gets the D-Xylitol.
10. osmophilic yeast bacterial classification that transforming glucose is an arabitol, it is characterized in that: said bacterial classification is Ao Mo Kodak yeast (Kodamaea ohmeri) TL-1123 CGMCC NO.6197.
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| CN106661540A (en) * | 2014-06-18 | 2017-05-10 | 罗盖特兄弟公司 | Production of xylitol from glucose by a recombinant strain |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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|---|---|---|---|---|
| US5238826A (en) * | 1989-06-16 | 1993-08-24 | Roquette Freres | Process for manufacturing xylose |
| CN1284564A (en) * | 1999-06-24 | 2001-02-21 | 味之素株式会社 | Method for production of D-arabitol, D-xyloketose and xylitol |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5238826A (en) * | 1989-06-16 | 1993-08-24 | Roquette Freres | Process for manufacturing xylose |
| CN1284564A (en) * | 1999-06-24 | 2001-02-21 | 味之素株式会社 | Method for production of D-arabitol, D-xyloketose and xylitol |
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| CN106661540A (en) * | 2014-06-18 | 2017-05-10 | 罗盖特兄弟公司 | Production of xylitol from glucose by a recombinant strain |
| CN106661540B (en) * | 2014-06-18 | 2021-02-09 | 罗盖特兄弟公司 | Production of xylitol from glucose by recombinant strains |
| US10759727B2 (en) | 2016-02-19 | 2020-09-01 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
| US11840500B2 (en) | 2016-02-19 | 2023-12-12 | Intercontinental Great Brands Llc | Processes to create multiple value streams from biomass sources |
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