CN1264853C - Method for extracting D-ribose crystal from fermented broth - Google Patents
Method for extracting D-ribose crystal from fermented broth Download PDFInfo
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Abstract
The present invention relates to a method for directly extracting a D-ribose crystal from fermentation liquor, which comprises: the fermentation liquor is treated with a flocculating agent or an adsorbing agent; flocculus precipitates or the adsorbing agent is separated; column chromatography is performed on clear filtrate; resin column effluent is concentrated; D-ribose is crystallized, separated, dried and crystallized. The present invention applying the methods of adsorption sedimentation after flocculation or acidification, etc. into the pretreatment of the fermentation liquor of the ribose intensifies the pretreatment procedure of the fermentation liquor, thoroughly removes mycoprotein and other impurities in the fermentation liquor and omits the procedures of concentration, alcohol settling, impurity removal, etc. of fermentation filter liquor not thoroughly purified in the prior art. The present invention has the advantages of extraction process simplification and product quality improvement.
Description
(1) technical field
The present invention relates to a kind of D-ribose crystalline method of from fermented liquid, directly extracting.
(2) background technology
D-ribose is the important composition composition of genetic material-nucleic acid in the organism, is again the metabolic important participant of organism self-energy, is in hub site in nucleosides material, protein, metabolism of fat, has the important physical function.
Industrial, D-ribose is vitamins B
2Synthesis material, can be used for the synthetic of antiviral, antitumor drug again.In recent years; along with of the widespread use of D-ribose at medical health field; particularly it is at the treatment myocardial ischaemia with prevent and treat exploitation because of new purposes of aspect health care such as muscle rigidity that motion causes, sore muscles; D-ribose more and more causes people's attention, and the various countries scientist falls over each other and is devoted to set up the preparation method of the D-ribose that is fit to the big production of mass-producing.
The preparation method of D-ribose comprises methods such as extraction separation, chemosynthesis and microbial fermentation from crude substance.Extraction separation D-ribose from natural goods, leaching process is numerous and diverse, manufacturing cost is high, is not suitable for large-scale production.With regard to synthesis method, people have successively attempted from D-pectinose (Gehrke and Aichner, 1927), maltonic acid (Steiger, 1936), D-glucose (Korczynski A et al.1979), L-L-glutamic acid and D-wood sugar (Lacourt Gadras, et al.1992) synthetic D-ribose, before 20th century the eighties, the main both at home and abroad suitability for industrialized production of carrying out D-ribose by glucose chemistry synthesis method that adopts, promptly by glucose through oxidation, displacement, transform, displacement again, acidifying, lactonize, seven steps reaction such as reduction makes D-ribose, this method is the step complexity not only, equipment used is many, yield is low, the manufacturing cost height, and its related tribute electrode reduction reaction also can cause serious environmental to pollute.After entering 20th century the nineties, people bring into use Production by Microorganism Fermentation D-ribose, and fermentation method carries out at normal temperatures and pressures, and the starting material wide material sources have been avoided the environmental pollution of chemical synthesis again, are called as the optimal best method of producing D-ribose.What extensively adopt in the fermentative Production is the microorganism of bacillus.
Fermentative Production D-ribose comprises fermentation and extracts two stages of crystallization that it is most important for the finished product yield, product quality and production cost to extract crystallisation stage.At present industrial from D-ribose fermented liquid extraction separation ribose crystalline method mainly contain two kinds, a kind of is to form riboside earlier by chemosynthesis reaction, such as forming α-aniline-N-D-pyrans or ribofuranose glucoside,, carry out the crystalline method more then through hydrolysis; Another kind is directly to extract D-ribose crystalline method from fermented liquid.Before a kind of method, not only the processing step complexity, yield is low, cost is high, and uses virose organic substance in the manufacturing processed, can cause environmental pollution inevitably.And second kind directly extracted D-ribose crystalline method, the incomparable advantage of a kind of method before having undoubtedly from fermented liquid.
Day disclosure special permission clear 51-91388 of communique and United States Patent (USP) 4,904,587 (1990) disclose a kind of D-ribose crystalline method of directly extracting from fermented liquid, this method is the thalline in centrifuging removal fermented liquid at first, concentrate ferment filtrate then to 1/2 of original volume, and after the Partial Protein and impurity in a certain amount of ethanol sedimentation fermented liquid, with clear liquid by cationic, anionic exchange resin desalination, activated carbon column decoloring after, concentrated, crystallization obtain D-ribose product.When adopting the crystallization of method for preparing D-ribose, fermented liquid also contains great amount of soluble albumen, organic-inorganic impurity and various ion behind centrifugal removal thalline and solid impurity, and wherein calcium ion content is high; Then ferment filtrate is concentrated into half of original volume, and adds a certain amount of ethanol, precipitation is removed Partial Protein and other alcohol insoluble matter impurity wherein.Owing to contain a large amount of calcium ions in the fermented liquid, in concentration process, not only can form very thick one deck calcification layer, be attached to the thickening equipment surface, be difficult to remove, and can produce a large amount of furfural class materials and other pigment, all can increase difficulty of post-processing and influence ribose crystalline quality, in addition, in scale operation, use ethanol sedimentation albumen and other impurity, use a large amount of ethanol, but can't reclaim.
Disclose the D-ribose fermentation that utilizes a bacillus subtilis and the purifying method of fermented liquid thereof among the Chinese patent CN 1122833A (1996), but the purification of fermented liquid is only accomplished till the D-ribose syrup.In another part Chinese patent CN 1234404A (1999) prospectus, a kind of method of utilizing ion exchange resin separating D-ribose from fermented liquid is disclosed, utilize ion exchange resin to passing through the fermented liquid behind the centrifugal removal thalline but only relate to, carry out the method for edulcoration purification, Special attention will be given to be that three types resin is comprised storng-acid cation exchange resin, weak base anion-exchange resin, weakly acidic cation-exchange resin is united use, it is through from the fermented liquid behind the removal thalline of D-ribose pure in the post liquid before for upper prop crossed of handing over post, yield can be up to more than 95%, and only mention and to pass through the post liquid excessively of ion exchange resin column through after concentrating, can obtain the crystallization of purity more than 98%, but all not mentioned to problems such as the crystallization method of D-ribose and crystallization yields.Since fermented liquid last from the friendship post before, just through centrifugal removal thalline, also contain a large amount of soluble proteinss, organic or inorganic impurity etc., although ion exchange resin is weak base anion-exchange resin especially, except effect with adsorbed ion, also have the ability of other impurity of absorption, but for the fermented liquid that contains great amount of soluble protein ingredient complexity,, be to be difficult to completely only with the Impurity removal in the fermented liquid by ion exchange resin.Such as it also can contain a large amount of alcohol insoluble matter impurity in handing over liquid, and this purifies for follow-up crystalline, causes very big difficulty.
Therefore, industrial present urgent need is set up and is a kind ofly directly extracted the crystallization of D-ribose, technology advantages of simple from fermented liquid, is suitable for making at an easy rate on a large scale D-ribose crystalline method.
(3) summary of the invention
[problem that will solve]
Purpose of the present invention provides a kind of D-ribose crystalline novel method of directly extracting from the ribose fermented liquid.
[technical scheme]
The contriver has invented a kind of new D-ribose crystalline method of directly extracting by further investigation from fermented liquid.This method may further comprise the steps:
1) with flocculation agent or sorbent treatment fermented liquid;
2) separate floss precipitation or sorbent material;
3) cleaner liquid is carried out column chromatography;
4) the concentrated resin post is crossed flow liquid;
5) make the crystallization of D-ribose;
6) separate also drying crystalline.
Above method the 1st) and 2) step in fact is that fermented liquid is carried out pre-treatment, purpose is somatic cells, albumen, organic macromolecule and other the residual solid impurity of removing in the fermented liquid.Wherein flocculation agent can be an organic floculant, as chitin, chitosan, and also available inorganic flocculating agent, as poly aluminium chloride, flocculation agent can use separately, but that they and coagulant aids (as hydro-polyacrylamide) are used effect is better.
In above method, handling fermented liquid with flocculation agent is realized by following process: regulate the fermented liquid potential of hydrogen to slightly acidic, neutrality or weakly alkaline, promptly regulate fermented liquid pH value in certain scope, with the flocculation agent newly prepared under agitation, slowly add in the fermented liquid, after stirring, leave standstill, when treating that the suspension cohesion forms a large amount of flosss and begins sedimentation, stir once more, coagulant aids is slowly added, after stirring, leave standstill.
Sorbent material can be to have synergistic yellow prussiate of potash and zinc sulfate, or calcium chloride and Sodium phosphate dibasic.Acidifying can be used oxalic acid, also available sulfuric acid.Realize by following process with the sorbent treatment fermented liquid: use the steam heating fermented liquid, add oxalic acid and regulate the fermented liquid potential of hydrogen to strongly-acid, promptly regulate fermented liquid pH value in 1.5 to 3.5 scopes, add yellow prussiate of potash and zinc sulfate successively, perhaps calcium chloride and Sodium phosphate dibasic are warming up to 60～85 ℃ of insulations 10 to 30 minutes.
In above method, the sedimentary method of wherein said separation floss comprise siphon, press filtration and centrifugal in one or more.
In above method, wherein saidly cleaner liquid is carried out column chromatography comprise cleaner liquid is carried out cation exchange resin column chromatography, anion-exchange resin column chromatography, macroporous adsorption resin chromatography and decolorizing resin column chromatography successively; Perhaps cleaner liquid is carried out cation exchange resin column chromatography, anion-exchange resin column chromatography, amphoteric resin column chromatography and decolorizing resin column chromatography successively.Can adhesion protein and polymeric adsorbent of pigment, the ion exchange resin that can remove zwitterion, the activated carbon decolorizing resin that can decolour etc. may be used to method of the present invention.Ion exchange resin both can use weakly acidic cation-exchange resin, weak base anion-exchange resin, also can use storng-acid cation exchange resin and strongly basic anion exchange resin, wherein preferably storng-acid cation exchange resin and weak base anion-exchange resin; Macroporous resin adsorption can be a nonpolar macroporous adsorption resin, also can be the macroporous adsorbent resin of middle polarity.They be used better effects if, preferred mode is storng-acid cation exchange resin, weak base anion-exchange resin, middle polarity macroporous adsorbent resin, activated carbon decolorizing resin are used, or storng-acid cation exchange resin, weak base anion-exchange resin, amphoteric resin, decolorizing resin are used.The resin that can be used for the inventive method, for example, Zeo-karb has 732,001 * 7,001 * 8, the AmberliteIR-120 Zeo-karb; Anionite-exchange resin has D315, D330, D335, AmberliteIR-63 anionite-exchange resin; Macroporous adsorbent resin has HZ-803, SD300, SD301, SD302; Amphoteric resin has the TP-1 resin; Decolorizing resin has D730, D750, D314, granulated active carbon decolorizing resin.
In above method, the method that wherein said concentrated resin post is crossed flow liquid refers to the vacuum decompression method of enrichment, and the temperature of vacuum concentration is between 35～40 ℃.
In above method, the wherein said D-of making ribose crystalline method is realized by following process: press the volume of 2～4 times of heavy syrups, add dehydrated alcohol, slightly heated is fully miscible, adds small amount of seeds and cooling.Tc is generally 5～30 ℃, mass crystallization can occur in 10～18 hours.
In above method, separate and the method for drying crystalline comprises that those of ordinary skills are known and anyly crystal is separated with mother liquor and can make crystal exsiccant method.Operable method for example, with the method for centrifugation or vacuum filtration, obtain wet crystallization after, the crystallization of will wetting of available double-cone vacuum drier was dried by the fire 3～4 hours under-0.095Mpa, 55 ℃ of conditions, can obtain D-ribose finished product.
Above-described step can be carried out separately, also can carry out or repeat in conjunction with carrying out, can also putting upside down.
Method of the present invention can be used for all fermented liquids that can produce the microorganism of D-ribose, the fermented liquid that preferably is used for bacillus micro-organism, for example, subtilis (Bacillussubtilis) and bacillus pumilus (Bacillus pumilus) fermented liquid.
Regulation according to patent law of china and its detailed rules for the implementation, bacillus pumilus P8069 of the present invention and subtilis S6023 are deposited in China Microbial Culture Preservation Commission common micro-organisms center (CGMCC) on December 5th, 2002, and preserving number is respectively CGMCC 0845 and CGMCC 0846.
[beneficial effect]
The present invention will flocculate or acidifying after method such as adsorption and sedimentation apply in the pre-treatment of ribose fermented liquid, removed a large amount of albumen and other impurity in the fermented liquid more up hill and dale, saved in the prior art without the concentrating of the ferment filtrate of thorough purifying, alcohol falls and operation such as removal of impurities, simplified greatly and from fermention medium, directly extracted D-ribose crystalline method, and improved quality product.The D-ribose crystallization that utilizes method of the present invention directly to extract from fermented liquid, a step total recovery can reach more than 60%, and mother liquor reclaims again crystallization yield and can reach more than 10%, utilizes this technology purification D-ribose crystalline total recovery to reach more than 70% like this.Detect through high pressure liquid chromatography, the D-ribose crystalline purity that is obtained reaches more than 99%.
(4) embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
From bacillus pumilus CGMCC 0845 broth extraction D-ribose crystallization
With glucose 1.0% (bulking value content w/v, down together), extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation bacillus pumilus CGMCC 0845 (the transketolase activity is 0 bacillus pumilus mutant strain) cultivated 24～36 hours in 36 ℃, after treating that lawn is grown well, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by sorbyl alcohol 2.0% corn steep liquor 2.0%, yeast extract paste 0.1%, dipotassium hydrogen phosphate 0.3%, potassium primary phosphate 0.1% is in the 10L seed culture medium (pH7.0) that sal epsom 0.05% is formed, cultivate after 18 hours for 36 ℃, access is by glucose 20.0% (w/v), corn steep liquor 2.6%, ammonium sulfate 0.7%, manganous sulfate 0.005% is in the 100L fermention medium that lime carbonate 2.2% (pH7.0) is formed.200 rev/mins of control mixing speed, 36 ℃ of culture temperature, tank pressure 0.50kg/cm
2, earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 20～25%; The fermentation middle and later periods is controlled pH6.3～6.5, dissolved oxygen 25～30%, and aeration-agitation was cultivated 46 hours, accumulation D-ribose 83.20g/L.
The flocculation of fermented liquid utilizes chitin as flocculation agent, and dosage is pressed the 350ppm of fermented liquid total amount, and as coagulant aids, dosage is pressed the 50ppm of fermented liquid total amount with hydro-polyacrylamide.The compound method of flocculation agent: with chitin sheet or meal, the ratio with 0.5～1.0% is dissolved in the dilute acid soln of 0.1～1.0mol; The compound method of coagulant aids: a coagulant aids particle shaped product is added to the water, stirs, adds one times of water with 0.1～0.2% ratio, treat that this coagulant aids material all immerses in the aqueous solution, promptly stop to stir, after leaving standstill 2～3 hours, solution becomes gets thickness, stirs this solution gently and gets final product to homogeneous transparent up and down.Flocculation process: fermented liquid 100L, transfer pH to 7.0～7.8, under agitation condition, slowly add in the fermented liquid freshly prepared flocculation agent, after adding, continue to stir 5 minutes, left standstill 15 minutes, when treating that the suspension cohesion forms a large amount of flosss and begins sedimentation, stir once more, coagulant aids is slowly added, continue to stir 5 minutes, left standstill 2 hours.Thalline, macromolecular substance and other impurity molecule in the fermented liquid condenses into floss and is settled down to the fermented liquid bottom, the clarification of upper strata filtrate at this moment.With the supernatant liquid sucking-off, lower floor's feed liquid is removed impurity with press filtration or centrifugal mode, obtains cleaner liquid with siphonage.Cleaner liquid is passed through 732 Zeo-karbs, D335 anionite-exchange resin, HZ-803 nonpolar macroporous adsorption resin, granulated active carbon decolorizing resin successively.(cm * cm) dress post amount is 30L to post bed specification 20 * 150.Collect resin column and cross flow liquid, under-0.095Mpa vacuum degree condition, 35～40 ℃, be evaporated to syrupy shape, add the ethanol of 2～4 times of amounts of syrup volume, slightly heated miscible evenly after, add small amount of seeds, be cooled to 5 ℃, 10～18 hours, the crystallization of D-ribose forms in a large number, centrifugally gets rid of filter, drying under reduced pressure gets D-ribose crystallization 51.15kg.Crystalline mother solution is by behind the concentrating under reduced pressure, and ethanol can reclaim through the smart stream of ethanol tower and reuse, and the D-ribose in the mother liquor can obtain D-ribose crystallization 8.37kg again by above-mentioned purifies and separates process.So altogether D-ribose crystallization 59.52kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.68, and specific optical rotation is-20.76 (in 4% the aqueous solution, 20 ℃).
Embodiment 2
From subtilis CGMCC 0846 broth extraction D-ribose crystallization
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation subtilis CGMCC 0846 (the transketolase activity is 0 bacillus subtilis mutant strain), cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be eggplant bottle slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by glucose 2.0% yeast extract paste 0.3%, corn steep liquor 0.05%, dipotassium hydrogen phosphate 0.3%, potassium primary phosphate 0.1% is in the 10L seed culture medium that sal epsom 0.05% (pH7.0) is formed, cultivate after 18 hours for 36 ℃, access is by glucose 20.0%, corn steep liquor 2.6%, ammonium sulfate 0.7%, manganous sulfate 0.005% is in the 100L fermention medium (pH7.0) that lime carbonate 2.2% is formed.220 rev/mins of control mixing speed, 36 ℃ of culture temperature.220 rev/mins of control mixing speed, 36 ℃ of culture temperature.Earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 18～22%; The fermentation middle and later periods is controlled pH6.0～6.2, dissolved oxygen 20～25%; At 36 ℃, 0.60kg/cm
2Condition under, aeration-agitation was cultivated 42 hours, accumulation D-ribose 83.60g/L.
The flocculation of fermented liquid utilizes poly aluminium chloride and hydro-polyacrylamide, dosage is respectively the 300ppm and the 100ppm of fermented liquid total amount, poly aluminium chloride is made into 20% solution, and the concentration of hydro-polyacrylamide 0.2% is formulated by the compound method identical with example 1.Flocculation process: fermented liquid 100L, transfer pH to 6.7～7.5, with fresh preparation poly aluminium chloride and hydro-polyacrylamide solution successively under agitation condition, slowly add in the fermented liquid, after adding, continue to stir 5 minutes, left standstill 2 hours, thalline, macromolecular substance and other impurity molecule in the fermented liquid condenses into floss and is settled down to the fermented liquid bottom, the clarification of upper strata filtrate at this moment.With the supernatant liquid sucking-off, lower floor's feed liquid is removed impurity with press filtration or centrifugal mode, obtains cleaner liquid with siphonage.Cleaner liquid is passed through 732 Zeo-karbs, D335 anionite-exchange resin, TP-1 amphoteric resin, granulated active carbon decolorizing resin successively.Post bed specification 20 * 150cm, dress post amount is 30L.Collect resin column and cross flow liquid, under-0.095Mpa vacuum degree condition, 35～40 ℃, be evaporated to syrupy shape, add the ethanol of 2～4 times of amounts of syrup volume, slightly heated miscible evenly after, add small amount of seeds, be cooled to 5 ℃, 10～18 hours, the crystallization of D-ribose forms in a large number, centrifugal filter, the drying under reduced pressure acquisition D-ribose crystallization 51.25kg of getting rid of.Crystalline mother solution is by behind the concentrating under reduced pressure, and ethanol can reclaim through the smart stream of ethanol tower and reuse, and the D-ribose in the mother liquor can regain crystallization 8.39kg by above-mentioned purifies and separates process.So altogether D-ribose crystallization 59.64kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.66%, and specific optical rotation is-20.60 (in 4% the aqueous solution, 20 ℃).
Embodiment 3
From subtilis CGMCC 0846 broth extraction D-ribose crystallization
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation subtilis ATCC0846, cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in example 2 and form in the identical 10L seed culture medium, cultivate after 16 hours for 36 ℃, access is by glucose 20.0%, corn steep liquor 2.6, ammonium sulfate 0.7, manganous sulfate 0.005, lime carbonate 2.2% is in the 100L fermention medium of composition.250 rev/mins of control mixing speed, 36 ℃ of culture temperature.Earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 25～30%; The fermentation middle and later periods is controlled pH6.0～6.2, dissolved oxygen 30～35%; At 36 ℃, 0.7kg/cm
2Condition under, aeration-agitation was cultivated 45 hours, accumulation D-ribose 80.5g/L.
Fermented liquid 100L, with being steam heated to 45～50 ℃, slowly add oxalic acid, transfer pH to 1.5～3.5,0.1～0.2% the amount of pressing fermentating liquid volume then successively adds yellow prussiate of potash, the amount by 0.05～0.1% adds zinc sulfate, be warming up to 70～75 ℃ of insulations 20 minutes, remove impurity, obtain cleaner liquid with press filtration or centrifugal mode.Cleaner liquid is passed through 732 Zeo-karbs, D314 anionite-exchange resin, HZ-803 nonpolar macroporous adsorption resin, granulated active carbon resin successively.(cm * cm), dress post amount is 30L to post bed specification 20 * 150.Collect resin column and cross flow liquid, under-0.095Mpa vacuum degree condition, 35～40 ℃, be evaporated to syrupy shape, add the ethanol of 2～4 times of amounts of syrup volume, slightly heated miscible evenly after, add small amount of seeds, be cooled to 5 ℃, 10～18 hours, the crystallization of D-ribose forms in a large number, centrifugal filter, the drying under reduced pressure acquisition D-ribose crystallization 49.80kg of getting rid of.Crystalline mother solution is by behind the concentrating under reduced pressure, and ethanol can reclaim through the smart stream of ethanol tower and reuse, and the D-ribose in the mother liquor can regain crystallization 8.70kg by above-mentioned purifies and separates process.So altogether crystallization 58.50kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.80%, and specific optical rotation is-20.57 (in 4% the aqueous solution, 20 ℃).
Embodiment 4
From the crystallization of bacillus pumilus ATCC31093 broth extraction D-ribose
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation bacillus pumilus ATCC31093, cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by sorbyl alcohol 2.0%, corn steep liquor 2.0%, dipotassium hydrogen phosphate 0.3%, potassium primary phosphate 0.1%, in the 10L seed culture medium that each 100ug/ml of tyrosine and phenylalanine (pH) forms, cultivate after 16 hours for 36 ℃, access is by glucose 20.0%, dry yeast 1.2%, ammonium sulfate 0.6%, manganous sulfate 0.005%, lime carbonate 2.2% is in the 100L fermention medium (pH7.0) that each 50 μ g/ml of tryptophane, tyrosine and phenylalanine form.260 rev/mins of mixing speed of control, earlier fermentation 0～16 hour, control pH6.5～7.0, dissolved oxygen 20～25%, air flow quantity 1: 0.15～1: 0.25; Fermentation middle and later periods control pH6.3～6.5, dissolved oxygen 25～30%, air flow quantity 1: 0.25～1: 0.70; At 37 ℃, 0.50kg/cm
2Condition under, aeration-agitation was cultivated 46 hours, accumulation D-ribose 81.70g/L.
Press the extracting and purifying method of example 1, directly from fermented liquid, obtain D-ribose crystallization 50.27kg, from mother liquor, reclaim purifying and obtain D-ribose crystallization 8.26kg, altogether D-ribose crystallization 58.53kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.35%, and specific optical rotation is-20.25 (in 4% the aqueous solution, 20 ℃).
Embodiment 5
From the crystallization of subtilis IFO13621 broth extraction D-ribose
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation subtilis IFO13621, cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by sorbyl alcohol 2.0%, corn steep liquor 2.0%, dipotassium hydrogen phosphate 0.3%, potassium primary phosphate 0.1%, in the 10L seed culture medium that each 100ug/ml of tyrosine and phenylalanine (pH) (pH7.0) forms, cultivate after 18 hours for 36 ℃, access is by glucose 20.0%, dry yeast 1.0%, ammonium sulfate 0.6%, manganous sulfate 0.005%, lime carbonate 2.2% is in the 100L fermention medium (pH7.0) that each 50 μ g/ml of tryptophane, tyrosine and phenylalanine form.280 rev/mins of mixing speed of control are by at earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 20～25%; The fermentation middle and later periods is controlled pH6.1～6.3, dissolved oxygen 25～30%; At 36 ℃, 0.50kg/cm
2Condition under, aeration-agitation was cultivated 46 hours, accumulation D-ribose 81.50g/L.
Press the extracting and purifying method of example 2, directly from fermented liquid, obtain D-ribose crystallization 49.10kg and from mother liquor, reclaims purifying and obtain D-ribose crystallization 8.27kg, common D-ribose crystallization 57.37kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.27%, and specific optical rotation is-20.48 (in 4% the aqueous solution, 20 ℃).
Embodiment 6
From the crystallization of subtilis S1907 broth extraction D-ribose
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation variant S1907 (the transketolase activity is 0 bacillus subtilis mutant strain), cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by sorbyl alcohol 2.0% corn steep liquor 2.0%, yeast extract paste 0.1%, dipotassium hydrogen phosphate 0.3%, potassium primary phosphate 0.1%, sal epsom 0.05% in the 10L seed culture medium that pH7.0 (pH7.0) forms, is cultivated after 18 hours for 36 ℃, access is by glucose 20.0%, corn steep liquor 2.6%, dry yeast 0.10%, ammonium sulfate 0.7%, manganous sulfate 0.005% is in the 100L fermention medium that lime carbonate 2.2% (pH7.0) is formed.220 rev/mins of mixing speed of control, earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 20～25%; The fermentation middle and later periods is controlled pH6.2～6.4, dissolved oxygen 25～30%; At 37 ℃, 0.50kg/cm
2Condition under, aeration-agitation was cultivated 46 hours, accumulation D-ribose 80.2g/L.
Press the extracting and purifying method of embodiment 3, directly from fermented liquid, obtain D-ribose crystallization 49.65kg, from mother liquor, reclaim purifying and obtain D-ribose crystallization 8.57kg, altogether D-ribose crystallization 58.22kg.Through high-pressure liquid phase chromatogram therapy determining D-ribose crystallization purity is 99.95%, and specific optical rotation is-20.80 (in 4% the aqueous solution, 20 ℃).
Embodiment 7
From the crystallization of bacillus pumilus P8009 broth extraction D-ribose
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation bacillus pumilus P8009 (the transketolase activity is 0 bacillus pumilus mutant strain), cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by glucose 2.0% corn steep liquor 2.0%, yeast extract paste 0.05%, dipotassium hydrogen phosphate 0.3%, in the 10L seed culture medium that potassium primary phosphate 0.1%, sal epsom 0.05% (pH7.0) are formed, cultivate after 16 hours for 36 ℃, insert by glucose 20.0%, corn steep liquor 2.5%, ammonium sulfate 0.6%, manganous sulfate 0.005% is in the 100L fermention medium (pH7.0) that lime carbonate 2.2% is formed.220 rev/mins of control mixing speed, earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 20～25%; The fermentation middle and later periods is controlled pH6.1～6.3, dissolved oxygen 25～30%; At 36 ℃, 0.50kg/cm
2Condition under, aeration-agitation was cultivated 46 hours, accumulation D-ribose 79.60g/L.
Press the extracting and purifying method of embodiment 1, directly from fermented liquid, obtain D-ribose crystallization 48.10kg, from mother liquor, reclaim purifying and obtain D-ribose crystallization 8.25kg, altogether D-ribose crystallization 56.35kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.65%, and specific optical rotation is-20.17 (in 4% the aqueous solution, 20 ℃).
Embodiment 8
From the crystallization of subtilis S6057 broth extraction D-ribose
With glucose 1.0%, extractum carnis 1.4%, peptone 0.7%, yeast extract paste 0.4%, agar 2.0% is eggplant bottle slant medium (pH7.0), by microorganism routine operation method bevel, in 36 ℃ of constant temperature culture 48 hours, when observing no varied bacteria growing, inoculation subtilis S6057, cultivated 24～36 hours in 36 ℃, treat lawn grow good after, be slant strains.Long good eggplant bottle slant strains is made bacteria suspension with stroke-physiological saline solution, be inoculated in by glucose 2.0% yeast extract paste 0.3%, corn steep liquor 0.05%, dipotassium hydrogen phosphate 0.3%, potassium primary phosphate 0.1% is in the 10L seed culture medium (pH7.0) that sal epsom 0.05% (pH7.0) is formed, cultivate after 18 hours for 36 ℃, access is by glucose 20.0%, corn steep liquor 2.6%, ammonium sulfate 0.7%, manganous sulfate 0.005% is in the 100L fermention medium (pH7.0) that lime carbonate 2.2% is formed.260 rev/mins of mixing speed of control, earlier fermentation 0～18 hour, control pH6.5～7.0, dissolved oxygen 20～25%; The fermentation middle and later periods is controlled pH6.0～6.2, dissolved oxygen 25～30%; At 37 ℃, 0.50kg/cm
2Condition under, aeration-agitation was cultivated 46 hours, accumulation D-ribose 81.5g/L.
Press the extracting and purifying method of embodiment 1, directly from fermented liquid, obtain D-ribose crystallization 48.95kg, from mother liquor, reclaim purifying and obtain D-ribose crystallization 8.39kg, altogether D-ribose crystallization 57.34kg.Through high-pressure liquid chromatography D-ribose crystallization purity is 99.75%, and specific optical rotation is-20.32 (in 4% the aqueous solution, 20 ℃).
Claims (4)
1. one kind is extracted D-ribose crystalline method from fermented liquid, and this method may further comprise the steps:
1) with flocculation agent or sorbent treatment fermented liquid
Wherein said flocculation agent is one or more in organic floculant chitin, chitosan, the inorganic flocculating agent poly aluminium chloride, or the mixture of they and coagulant aids hydro-polyacrylamide.
Wherein saidly handle fermented liquid with flocculation agent and is realized by following process: adjusting fermented liquid potential of hydrogen is to slightly acidic, neutrality or weakly alkaline, with newly the preparation flocculation agent under agitation, slowly add in the fermented liquid, stir, leave standstill, make the suspension cohesion, form a large amount of flosss, through sedimentation and clear liquid layering; If unite use with coagulant aids, when floss begins to form in a large number, stir once more, coagulant aids is slowly added, stir, leave standstill;
Wherein saidly realize by following process: use the steam heating fermented liquid, slowly add acid and regulate the fermented liquid potential of hydrogen, add sorbent material successively, be warming up to 70～75 ℃ of insulations 10 to 30 minutes to strongly-acid with the sorbent treatment fermented liquid;
2) separate floss precipitation or sorbent material
The method of wherein said separation floss precipitation or sorbent material comprise siphon, press filtration and centrifugal in one or more;
3) cleaner liquid is carried out column chromatography
Wherein said to cleaner liquid carry out column chromatography comprise to cleaner liquid carry out successively that cation exchange resin layer is analysed, anion exchange resin layer is analysed, macroporous adsorption resin chromatography and activated carbon decolorizing resin chromatography; Perhaps to cleaner liquid carry out successively that cation exchange resin layer is analysed, anion exchange resin layer is analysed, amphoteric resin chromatography and decolorizing resin chromatography;
4) the concentrated resin post is crossed flow liquid
The method that wherein said concentrated resin post is crossed flow liquid is a concentrating under reduced pressure under 35 ℃ of-40 ℃ of conditions;
5) make the crystallization of D-ribose;
6) separate also drying crystalline.
2. according to the process of claim 1 wherein that said sorbent material is yellow prussiate of potash and zinc sulfate, or calcium chloride and Sodium phosphate dibasic.
3. according to the process of claim 1 wherein that said Zeo-karb comprises 732,001 * 7,001 * 8, the AmberliteIR-120 Zeo-karb; Anionite-exchange resin comprises D315, D330, D335, AmberliteIR-63 anionite-exchange resin; Macroporous adsorbent resin comprises HZ-803, SD300, SD301, SD302; Amphoteric resin TP-1 resin; Decolorizing resin comprises D730, D750, D314, granulated active carbon decolorizing resin.
4. according to the process of claim 1 wherein what the said D-of making ribose crystalline method was realized by following process: add ethanol, slightly heated adds small amount of seeds and cooling.
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| CN101475970B (en) * | 2008-01-04 | 2013-06-12 | 上海希迪制药有限公司 | Method for producing crystal D-ribose |
| CN102241706B (en) * | 2010-12-31 | 2014-04-09 | 三达膜科技（厦门）有限公司 | D-ribose purification and separation method |
| CN103113423B (en) * | 2013-03-15 | 2016-06-01 | 江西诚志生物工程有限公司 | A kind of method adopting ion-exchange and membrane separation technique to extract D-ribose from fermented liquid |
| CN103145771B (en) * | 2013-03-15 | 2016-08-03 | 江西诚志生物工程有限公司 | A kind of method using ultrafiltration and ion exchange technique to extract D-ribose from fermentation liquid |
| CN103739634B (en) * | 2014-01-14 | 2016-01-20 | 山东省食品发酵工业研究设计院 | A kind of method for implementing drying containing wet D-ribose crystallization |
| CN104447889A (en) * | 2014-12-18 | 2015-03-25 | 郑州拓洋生物工程有限公司 | Preparation method of high-purity D-ribose |
| CN107034318A (en) * | 2016-11-30 | 2017-08-11 | 山东福田药业有限公司 | A kind of processing method of xylose hydrolysis fluid |
| CN106755614A (en) * | 2016-11-30 | 2017-05-31 | 山东福田药业有限公司 | A kind of method of xylose purity in raising xylose hydrolysis fluid |
| CN109775911A (en) * | 2017-11-13 | 2019-05-21 | 秦皇岛华恒生物工程有限公司 | A method of l-Alanine mother liquor is handled using Simulation moving bed |
| CN115581277A (en) * | 2022-07-01 | 2023-01-10 | 江西省德兴市百勤异Vc钠有限公司 | Cadmium reduction treatment method for cadmium-polluted rice |
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Assignee: JIANGXI CHENGZHI BIOLOGICAL ENGINEERING Co.,Ltd. Assignor: Chengzhi Life Sci. & Tech. Co.,Ltd. Contract record no.: 2011360000083 Denomination of invention: Method for extracting D-ribose crystal from fermented broth Granted publication date: 20060719 License type: Exclusive License Open date: 20040630 Record date: 20110901 |
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