CN101918576A - A preparation method of disodium 5'-inosinate by using crystallization process - Google Patents
A preparation method of disodium 5'-inosinate by using crystallization process Download PDFInfo
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- CN101918576A CN101918576A CN200880122759XA CN200880122759A CN101918576A CN 101918576 A CN101918576 A CN 101918576A CN 200880122759X A CN200880122759X A CN 200880122759XA CN 200880122759 A CN200880122759 A CN 200880122759A CN 101918576 A CN101918576 A CN 101918576A
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Abstract
The present invention relates to a preparation method of disodkm 5'-inosinate(IMP2Na) from 5'-inosinic acid(IMP) fermentation liquid produced by the fermentation with microorganism. By the present invention, it can provide effect to reduce waste water compared to ion exchanged resin method and to produce disodium 5'-inosinate of high purity only by the control of temperature and the use of small amount of organic solvent.
Description
Technical field
The present invention relates to a kind of method by crystallisation process preparation 5 '-inosine acid disodium (IMP2Na), in more detail, relate to a kind of in purifying 5 '-inosine acid disodium process from t-inosinic acid (IMP) production bacterial strain fermentation liquor, replace existing ion exchange resin method by crystallisation process, to prepare the method for 5 '-inosine acid disodium.
Promptly, the present invention relates to a kind of under the situation of the identical 5 '-inosine acid disodium crystal yield of maintenance, increase the method for 5 '-inosine acid disodium crystal purity, simultaneously, by improving the crystallisation process of the used organic solvent of prior art, according to the distinctive temperature of performance, generation crystalline time point and vegetative point, control solubleness, reduced the consumption of organic solvent significantly.
Background technology
The purine derivatized nucleotide is as 5 '-inosine acid disodium (IMP2Na), as the prevention of metabolism disorder and the starting material of medicine, growth stimulant and cancer therapy drug, also as foodstuff additive, as the important materials of seasonings in the foodstuffs industry.
5 '-inosine acid disodium, a kind of composition of seasonings, be from the natural t-inosinic acid (IMP) that is present in meat and the fish etc., prepare by several different methods (as fish and meat extraction method, RNA enzyme liberating method, microbe fermentation method, fermentation and synthetic integrated process or RNA chemical degradation and synthetic integrated process).
In a kind of preparation method as the raw-material t-inosinic acid for preparing 5 '-inosine acid disodium, we have carried out the big quantity research by microbe fermentation method preparation 5 '-inosine acid disodium, and have been awarded multinomial patent.
But, because the product of aforesaid method contains the material of following kind: coloring matter, sugar, amino acid, organic acid, inorganic salt etc., therefore, must carry out purifying and remove wherein impurity, to obtain 5 '-inosine acid disodium with the quality that can be used for foodstuff additive to it.
Before, the method by ion-exchange resin purification 5 '-inosine acid disodium is by a plurality of stages, to use Zeo-karb and anionite-exchange resin separating organic substances and inorganic substance, to obtain highly purified t-inosinic acid solution; Concentrate then and crystallization t-inosinic acid solution, to obtain 5 '-inosine acid disodium crystal (Japanese Unexamined Patent Publication No 40-39715).But, make the purification process of spent ion exchange resin have the defective that produces a large amount of waste water.
Another that has overcome above-mentioned waste water related defects is forwardly in the method, disclose with mycothallus separate microorganism fermented liquid, and not spent ion exchange resin, concentrated supernatant then, then use hydrophilic organic solvent to carry out repeatedly crystalline method, obtain highly purified 5 '-inosine acid disodium.
As consideration to the patented technology of using aforesaid method, disclose method (korean patent application publication number 1987-0007197), preparation 5 '-inosine acid disodium and the 5 '-sodium guanylate mixed crystal of purifying 5 '-inosine acid disodium and 5 '-sodium guanylate mixture method (korean patent application publication number 1988-0010694), form 5 '-sodium guanylate crystalline method (korean patent application publication number 1993-0009984) and prepare the method (korean patent application publication number 2004-0018435) of 5 '-sodium guanylate and 5 '-inosine acid disodium mixed crystal.
But, problem is to separate with mycothallus and concentrate when microbial fermentation solution, and when using hydrophilic organic solvent to obtain 5 '-inosine acid disodium then from concentrated solution, the consumption of hydrophilic organic solvent is big, and because the adding of hydrophilic organic solvent, impurity also simultaneously has been extracted out.Also have, because extraction and the content of the t-inosinic acid that reduces can influence crystallisation process subsequently impurity the time, so be necessary the amount that joins by the organic solvent in the isolating fermented liquid of mycothallus is optimized.
Therefore, we have studied the method for purifying t-inosinic acid from microbial fermentation solution by paying close attention to the crystallisation process that uses hydrophilic organic solvent.Particularly, in order to address the above problem, we have repeated experiment and have improved crystalline output and purity, and minimize the amount of organic solvent, therefore, we have realized industrial available invention, compared with prior art, improve the purity of 5 '-inosine acid disodium, and under the situation of the consumption that reduces organic solvent, can keep its output constant.
Summary of the invention
The purpose of this invention is to provide the method for producing 5 '-inosine acid disodium, this method is used the microbial fermentation processes that can improve problems of the prior art, wherein, has reduced the amount of organic solvent significantly and has improved crystalline purity.
Realize purpose of the present invention by crystallisation process, set up optimized conditions, determined the purity of the 5 ' inosine acid disodium that obtains then by using based on the solubleness of the organic solvent of temperature and adding.
The method of preparation 5 ' provided by the invention-inosine acid disodium comprises: the first step, from the fermented liquid of t-inosinic acid production bacterial strain, separate and the removal mycothallus; In second step, concentrate described fermented liquid; In the 3rd step, the pH value of spissated fermented liquid is adjusted to 6-10; In the 4th step, the fermented liq behind the heat regulation pH is so that pre-crystalline 5 '-inosine acid disodium dissolves fully; In the 5th step, cooling contains dissolved 5 '-inosine acid disodium crystalline fermented liquid; In the 6th step, in cooled fermented liquid, add hydrophilic organic solvent, so that the crystallization of 5 '-inosine acid disodium.
Used microbial fermentation solution among the present invention, by inoculation t-inosinic acid production strain culturing, the bacterial strain of cultivating usefulness can use any bacterial strain that can produce t-inosinic acid, is preferably coryneform bacteria (Corynebacterium sp).Coryneform bacteria is preferably cultivated the t-inosinic acid that is used for producing at the about 5%-10% of fermented liquid.
The first step of the present invention can by be selected from by filter, any one method in centrifugal and the group that thermal treatment is formed carries out, preferably carry out with membrane filtering method.Those skilled in the art can control the condition of the membrane filtering method that separates mycothallus easily.For example, fermented liquid can be preheating to about 40-50 ℃, and intermembranous (TMP) pressure of 1.2-1.5atm can be used for this method.
Preferably, second the concentrated of step makes that t-inosinic acid concentration is 200-300g/L in the fermented liquid, most preferably, is 230-260g/L.
Preferably, the adjusting in the 3rd step makes the pH value of fermented liquid be 7.5-9.5.
Preferably, to make the temperature of fermented liquid be 55-75 ℃ in the heating in the 4th step.
The cooling in the 5th step is carried out in following condition: with uniform speed cooling, separate out naturally to induce heating back crystalline, at this, preferably made temperature be cooled to 10-30 ℃ in 3-6 hour.Based on this, preferred rate of cooling is 5-10 ℃/hour.
Preferably, the 6th step carried out in the following manner: when spissated fermented liquid is 20 ℃, add hydrophilic organic solvent.With the amount of 30-60 (v/v) % of fermented liquid total amount, add hydrophilic organic solvent.
In addition, described hydrophilic organic solvent can be lower alcohol, and one or more in particular methanol, ethanol, propyl alcohol, Virahol or their mixture most preferably are methyl alcohol, and the crystal shape that obtains in the methyl alcohol is suitable for solid-liquid most to be separated.
Hydrophilic organic solvent can use by dilute with water, but when thinning ratio increased, the add-on of organic solvent also increased, the solvent strength of industrial preferred 70-95 (v/v) %.
The present invention relates to prepare the method for 5 '-inosine acid disodium by crystallisation process, compare with ion-exchange-resin process, this method has reduced the generation of waste water, and, thereby the present invention is done well in foodstuffs industry only by controlled temperature and use a spot of organic solvent just can produce 5 ' high-purity-inosine acid disodium.
Description of drawings
Fig. 1 is the graphic representation that has shown in the concentrated broth based on the solubleness of the t-inosinic acid of methyl alcohol add-on %.
Fig. 2 is the graphic representation that has shown based on the crystalline supersaturation curve of the organic solvent methyl alcohol that adds.
Embodiment
Hereinafter, with reference to embodiment embodiments of the present invention and construction process thereof are described in more details, still, scope of the present invention is not limited to these embodiment.
The foundation of embodiment 1:5 '-inosine acid disodium crystallization condition
In order to set up the crystallization condition of 5 ' of optimization-inosine acid disodium, the solubleness of 5 '-inosine acid disodium is by temperature and organic solvent decision.The solubleness of 5 '-inosine acid disodium is measured with HPLC, when temperature is 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃, the concentration (%v/v) of organic solvent in total solution is regulated most 0-60% (Fig. 1).
The following method of supersaturation curve negotiating obtains: use online grain size analysis instrument, measure the solubleness of 5 ' inosine acid disodium solution in real attenuation liquid; Record becomes the point that increases suddenly based on karyomorphism temperature, under each organic solvent concentration (%v/v), and the point that karyomorphism becomes to suddenly disappear when temperature increases; Per minute is analyzed particulate number and particulate mean sizes and concentration etc.By experiment, by the organic solvent of temperature and adding, obtained the solubleness of 5 '-inosine acid disodium in the fermentation concentrated solution; Calculate percent crystallization in massecuite by cooling temperature, to seek suitable cooling temperature; And with compare by the cooling temperature that adds hydrophilic organic solvent and obtain from commitment.
The result as illustrated in fig. 1 and 2.Among the result, crystalline is separated out and is based on the supersaturation of t-inosinic acid in cultivating concentrated solution.Comparison as the supersaturation curve, different with the organic solvent of forcing to add, during cooling based on the concentration curve of the t-inosinic acid of time crystal formation in the actual concentrated solution and condition, stably appear in the solubility curve and the semi-stability district between the supersaturation curve of t-inosinic acid, therefore, can find that the crystalline size is owing to the inhibition of amorphous formation improves.
Embodiment 2: the crystallisation process of realizing cultivating concentrated solution by the crystallization of organic solvent
Based on the result of embodiment 1, from fermented liquid, prepare 5 '-inosine acid disodium as described below:
Step 1: the separating of the cultivation of bacterial strain and thalline
Produce corynebacterium glutamicum (Corynebacteriun glutamicum) CJM107 (KCCM-10227) bacterial strain of t-inosinic acid, be used for the present invention.
Inoculation is in contain 46g glucose, 30g fructose, 10g yeast extract, 18gKH in 1L
2PO
4, 42g K
2HPO
4, 6g urea, 10g MgSO
47H
2In the fermented liquid of O, 30 μ g vitamin Hs and 5mg vitamin, be exposed to pH 6-8, cultivating 140 hours in temperature 30-32 ℃ the 18L substratum, t-inosinic acid concentration reaches 5-10% in fermented liquid.The concentration of t-inosinic acid is measured with HPLC in the fermented liquid of cultivating.Beyond the coryneform bacteria other is used to produce the bacterial strain of t-inosinic acid arbitrarily, also can be used to realize same purpose.
Fermented liquid filters with film filter, to separate and to remove the mycothallus throw out, obtains filtering night.Filter and preferably to carry out when fermented liquid is heated to 50 ℃, to increase the separation efficiency of mycothallus, can predict filtration time and can shorten, because filtering segregation rate is higher during lower temperature in the time of 50 ℃, thereby productive rate also can increase.
But, it should be noted that when temperature to be increased to when being higher than 50 ℃, thermolysis can take place.From those of ordinary skills' angle, as filtration step, the centrifugal or heat treating method beyond the filter method also can use.
Step 2: the concentrating of filtering fermented liquid
Filtering fermented liquid concentrates with the vacuum tightness of 680mmHg and the drying under reduced pressure method of 55 ℃ of vessel temps, so that the concentration of t-inosinic acid becomes 230-260g/L.By t-inosinic acid concentration is concentrated into 200-300g/L, also can reach same result.
Step 3:pH value is regulated
35% HCl solution joins in the concentrated broth with the amount that adds 60mL among the 1L, to regulate pH value to 7.5.Because the output of separating out by crystalline is the highest, therefore the solubleness of 5 '-inosine acid disodium is minimum in the neutral pH zone, preferably regulates pH between neutrality and alkalescence, so that crystallization 5 ' from fermented liquid-inosine acid disodium.Therefore, can be 6-10 to regulate pH, to carry out this embodiment, preferably regulating pH with those skilled in the art's viewpoint is 7.5-9.5.250mL concentrated solution after the adjusting pH value is joined in the precipitation tube.
Step 4: heating
In order to stop concentrated solution to produce crystal in advance, the precipitation tube that will contain concentrated solution is heated to 60 ℃, is in the state that all crystal are all fused to keep liquid.With those skilled in the art's viewpoint, be heated to 55-75 ℃ and also can obtain same effect.
Step 5: the adding of organic solvent
Fermented liquid is heated to 60 ℃, and 95% (v/v) methanol aqueous solution of 350mL is joined in the fermented liquid in 5 hours with even flow rate, and keep 60 ℃ temperature.Except methyl alcohol, can use other hydrophilic organic solvent, as lower alcohol, be preferably in methyl alcohol, ethanol, propyl alcohol, the Virahol one or more, most preferably be methyl alcohol, the crystal shape that obtains in the methyl alcohol is suitable for solid-liquid most to be separated.
Hydrophilic organic solvent can use by dilute with water, but when thinning ratio increased, the add-on of organic solvent also increased, and the solvent strength of industrial preferred 70-100 (v/v) %.
Step 6: cooling separates with crystalline
After the adding of finishing methyl alcohol, temperature was reduced to 40 ℃ through 2 hours, separated out naturally with the mediation crystalline, used the basketry separator by the centrifugation crystal then.
The crystalline analysis that is separated to
The purity of 5 '-inosine acid disodium is determined with the HPLC quantitative method in the crystal that is separated to, after 105 ℃ of drying ovens descend dry 3 hours, measure water content, after under dissolution of crystals to 5% (w/v) concentration, the optical density instrument is measured the transmitance (T%) at 430nm place, and the crystalline gross weight that obtains is measured with electronic balance.The result is, the 5 '-inosine acid disodium purity that is separated to is 79%, and water content is 29%, and T% is 72.5, and gross weight is 67.5g.
Embodiment 3: carry out the crystallization of concentrated broth by crystallisation by cooling
Present embodiment 3 according to embodiment 2 in step 1 to 3 in same method carry out.Below explain the 4th step and later step.
Step 4: heating
Similar to embodiment 1, the temperature of crystallization precipitation tube is heated to 60 ℃, be in the state that all crystal are all fused to keep liquid.
Step 5: cooling
As the result of heating steps, liquid is not to produce the cooling of crystalline state.Liquid dropped to 20 ℃ uniform rate cooling in 5 hours, and the crystalline production rate is in 70% level of supernatant concentration.
Step 6: add organic solvent and isolation of crystalline
In order to extract the 5 '-inosine acid disodium that is in the crystallized form in the supernatant, 95% (v/v) methanol aqueous solution of 150mL was added in 2 hours.In the adding of methyl alcohol, the temperature of precipitation tube remains on 20 ℃, and is adding methyl alcohol post curing 30 minutes isolation of crystalline again.
The result is, isolating to 5 '-inosine acid disodium purity be 92.5%, water content is 31%, T% is 92.2, gross weight is 66.3g.By this result, aspect the raising crystal purity, it is effectively more than adding the organic solvent postcooling to determine to add organic solvent after cooling.
Embodiment 4 carries out the crystallization of concentrated broth by crystallisation by cooling (80% methyl alcohol)
Present embodiment 4 carries out with the method identical with embodiment 3 steps 1 to 5, and difference is to come comparative result by the concentration that changes the organic solvent that adds in the step 6.Hereinafter explained the content that step 6 is later.
The adding of step 6 organic solvent separates with crystalline
In order to extract the 5 '-inosine acid disodium that is in the crystallized form in the supernatant, 80% (v/v) methanol aqueous solution of 150mL was added in 3 hours.In the adding of methyl alcohol, the temperature of precipitation tube remains on 20 ℃, is adding methyl alcohol post curing 30 minutes isolation of crystalline more then.
The result is, isolating to 5 '-inosine acid disodium purity be 94.5%, water content is 30%, T% is 92.5, gross weight is 72.4g.
By this result, determine to access 5 ' inosine acid disodium with highest purity by the method for embodiment 4.
T-inosinic acid crystalline purity can depend on the concentration range of organic solvent, and therefore output is also inequality.As everyone knows, aspect crystal yield, separate out in the environment at crystal, it is favourable keeping the hydrophilic organic solvent adding rate with total liquid volume 30-40 (v/v) % equably.Can see that also surpassing under the situation of above-mentioned scope, impurity has also been separated out, and therefore becomes a reason that reduces crystal purity.
The content of the 5 '-inosine acid disodium that obtains among the embodiment 1-3 is 85-95%, and water content is lower than 28.5%.By the result of these embodiment, to compare with the method that adds organic solvent in the early stage crystal formation method (Korean Patent Application No. 10-2006-67035), the purity of 5 '-inosine acid disodium that the present invention obtains improves 10-20%.
As with the comparison of Korean Patent Application No. 10-2006-67035, the present invention has following effect: crystallisation process shortens to 1 time from 3 times, the amount of the organic solvent that adds reduces by crystallisation by cooling, and the content of 5 '-inosine acid disodium is increased to the level of 85-95%.These are summarised in the following table 1.
Table 1
| The add-on of organic solvent (v/v, %) | The content of #15 '-inosine acid disodium (%) | Crystal formation starting temperature (℃) | Cooling temperature (℃) | The adding temperature of organic solvent (℃) | |
| Existing method | 61 | 70 | 60 | 40 | 60 |
| Present method | 37 | 85 | 60 | 20 | 20 |
In the conventional method, organic solvent is 60 ℃ of addings, be cooled to 40 ℃ then, and in the crystallisation by cooling process that the present invention has improved, after being cooled to 20 ℃ from 60 ℃, a spot of organic solvent adds in the time of 20 ℃ to obtain crystal, makes the content of 5 '-inosine acid disodium be higher than prior art, thereby makes the present invention have positive effect.
Claims (9)
1. method for preparing 5 '-inosine acid disodium, wherein, this method comprises:
The first step is separated from the fermented liquid of t-inosinic acid production bacterial strain and the removal mycothallus;
In second step, concentrate described fermented liquid;
In the 3rd step, the pH value of spissated fermented liquid is adjusted to 6-10;
In the 4th step, the fermented liquid behind the heat regulation pH is so that pre-crystalline 5 '-inosine acid disodium dissolves fully;
In the 5th step, cooling contains dissolved 5 '-inosine acid disodium crystalline fermented liquid; And
In the 6th step, in cooled fermented liquid, add hydrophilic organic solvent, so that the crystallization of 5 '-inosine acid disodium.
2. the method for preparation 5 ' according to claim 1-inosine acid disodium, wherein, the concentration that is concentrated in t-inosinic acid in described second step stops during for 200-300g/L.
3. the method for preparation 5 ' according to claim 1-inosine acid disodium, wherein, the heating in described the 4th step is carried out under 55-75 ℃.
4. the method for preparation 5 ' according to claim 1-inosine acid disodium, wherein, the cooling in described the 5th step is carried out under the following conditions: with 5-10 ℃/hour speed, be cooled to 20-30 ℃.
5. the method for preparation 5 ' according to claim 1-inosine acid disodium, wherein, the adding in described the 6th step is carried out in the following manner: when described fermented liquid is 20 ℃, add described organic solvent.
6. the method for preparation 5 ' according to claim 1-inosine acid disodium, wherein, the adding in described the 6th step is carried out in the following manner: the amount with 30-40 (v/v) % of fermented liquid total amount adds described hydrophilic organic solvent.
7. the method for preparation 5 ' according to claim 1-inosine acid disodium, wherein, the hydrophilic organic solvent in described the 6th step is a lower alcohol.
8. the method for preparation 5 ' according to claim 7-inosine acid disodium, wherein, described lower alcohol is a methyl alcohol.
9. the method for preparation 5 ' according to claim 8-inosine acid disodium, wherein, the concentration of described methyl alcohol is 70-95 (v/v) %.
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| KR10-2007-0140740 | 2007-12-28 | ||
| KR1020070140740A KR100926253B1 (en) | 2007-12-28 | 2007-12-28 | A preparation method of disodium 5'-inosinate by using crystallization process |
| PCT/KR2008/007514 WO2009084836A2 (en) | 2007-12-28 | 2008-12-18 | A preparation method of disodium 5'-inosinate by using crystallization process |
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| CN114302888A (en) * | 2019-10-17 | 2022-04-08 | Cj第一制糖株式会社 | Method for separating disodium 5' -inosinate |
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| EP1411059A1 (en) * | 2001-07-26 | 2004-04-21 | Ajinomoto Co., Inc. | Method for producing mixed crystal of disodium 5'-guanylate and disodium 5'-inosinate |
| CN1508140A (en) * | 2002-11-22 | 2004-06-30 | 味之素株式会社 | Method for preparing purine derivative disodium nucleotate crystal and dealcoholing method |
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| KR890003253B1 (en) * | 1987-03-30 | 1989-08-31 | 제일제당 주식회사 | Process for crystallizing the mixture of gmp2na and imp2na |
| KR100721857B1 (en) * | 2006-07-20 | 2007-05-28 | 케이엔디티앤아이 주식회사 | THE SYSTEM OF OPERATION ON CRYSTALLIZATION USING Couette-Taylor REACTORS OVER IMP |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1411059A1 (en) * | 2001-07-26 | 2004-04-21 | Ajinomoto Co., Inc. | Method for producing mixed crystal of disodium 5'-guanylate and disodium 5'-inosinate |
| CN1527837A (en) * | 2001-07-26 | 2004-09-08 | ֮����ʽ���� | Method for producing mixed crystal of disodium 5'-guanylate and disodium 5'-inosinate |
| CN1508140A (en) * | 2002-11-22 | 2004-06-30 | 味之素株式会社 | Method for preparing purine derivative disodium nucleotate crystal and dealcoholing method |
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| CN114302888A (en) * | 2019-10-17 | 2022-04-08 | Cj第一制糖株式会社 | Method for separating disodium 5' -inosinate |
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| CN101918576B (en) | 2014-03-26 |
| KR20090072584A (en) | 2009-07-02 |
| WO2009084836A2 (en) | 2009-07-09 |
| KR100926253B1 (en) | 2009-11-12 |
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